Vaccine 32 (2014) 1869–1876

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Display of Eimeria tenella EtMic2 protein on the surface of Saccharomyces cerevisiae as a potential oral vaccine against chicken coccidiosis Hui Sun a,b , Longjiang Wang a,b , Tiantian Wang a,b , Jie Zhang a,b , Qing Liu a,b , Peipei Chen a,b , Zhengtao Chen a,b , Fangkun Wang a,b , Hongmei Li a,b , Yihong Xiao c , Xiaomin Zhao a,b,∗ a Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, 61 Daizong Street, Taian City, Shandong Province 271018, China b Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Agricultural University, 61 Daizong Street, Taian City, Shandong Province 271018, China c Department of Basic Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, 61 Daizong Street, Taian City, Shandong Province 271018, China

a r t i c l e

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Article history: Received 15 November 2013 Received in revised form 17 January 2014 Accepted 22 January 2014 Available online 11 February 2014 Keywords: Saccharomyces cerevisiae Cell surface display EtMic2 Live oral vaccine

a b s t r a c t S. cerevisiae is generally regarded as safe and benign organism and its surface display system may be used as a unique eukaryotic expression system that is suitable for expressing eukaryotic antigen. In addition to the convenience of vaccine delivery, the yeast cell wall has been shown to enhance the innate immunity when immunized with the yeast live oral vaccine. In the present study, we expressed the chicken coccidian E. tenella EtMic2, a microneme protein, on the surface of the S. cerevisiae and evaluated it as a potential oral vaccine for chicken against E. tenella challenge. The protective efficacy against a homologous challenge was evaluated by body weight gains, lesion scores and fecal oocyst shedding. The results showed that the live oral vaccine can improve weight gains, reduced cecal pathology and lower oocyst fecal shedding compared with non immunized controls. In addition, the yeast oral vaccine could stimulate humoral as well as cell mediate immune responses. These results suggested that EtMic2 displayed on the cell surface of S. cerevisiae could be used as potential live vaccine against chicken coccidiosis. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Chicken coccidiosis is caused by the apicomplexan protozoan Eimeria spp. and is a major parasitic disease affecting world-wide poultry industry. Eimeria tenella that parasitizes in chicken cecal epithelial cells is one of the most pathogenic species among the seven Eimeria species [1]. Although the chemoprophylaxis and live oocyst vaccines as primary method have been used to control chicken coccidiosis for many years, each approach has its own limitations and drawbacks. Firstly, due to the development of drug resistance against all of the anticoccidials developed thus far and more regulations and bans on the use of anticoccidial drugs, chemoprophylaxis strategies have become much more difficult and complex [2]. Secondly, despite the most live oocyst vaccines have been attenuated, they still can complete their endogenous development, therefore can affect the productivity of chickens, also it

∗ Corresponding author at: 61, Daizong Street, Taian City, Shandong Province 271018, China. Tel.: +86 0538 8249921; fax: +86 538 8249921. E-mail address: [email protected] (X. Zhao). 0264-410X/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2014.01.068

has the potency to reversion of virulence [3]. Therefore, novel approaches are urgently needed to control the chicken coccidiosis [4,5]. Recently, many attempts have been made to develop new types of coccidian vaccines such as the recombinant vaccine, the DNA vaccine, and the live oral vaccine as the effective alternative strategies to against coccidiosis [6–9]. Although these new type vaccines have some drawbacks including the limited immunogenicity and the lack of suitable immunoenhancing agents for use in the poultry industry [10–12], they showed a bright future for the coccidian vaccine development. Perhaps, the most prospective one is the live oral vaccine due to its easy to use [13]. The live oral vaccine is the vaccine that uses a microorganism as vehicle to express and deliver the designed antigen. The use of bacteria such as Salmonella, Shigella and Listeria as vehicles for vaccination against bacterium, virus and parasite infections has been reported [14–20]. As a live delivery system, yeast cellsurface display system is desirable in vaccine development due to the expressed proteins tightly linked to the cell wall glucan skeleton and the stable expression level [21,22]. Yeast is generally regarded as safe and benign organisms and the yeast cell wall components would increase growth rate [23] and modulate immunity of hosts

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Table 1 Experimental groups of chickens in immunization and challenge experiment. Groups

Immunogen (cells/chicken)

Immunization (days)

Challenged (day)

I (N = 20) II (N = 20) III (N = 20) IV (N = 20)

/ / HAO/pYSD (107 ) HAO/pYSD-EtMIC2 (107 )

/ / 2–21 2–21

/ 21 21 21

[24]. The use of S. cerevisiae as a vaccine vehicle has been used in veterinary field to protect hosts against viral diseases [25–28]. The use of S. cerevisiae as a vaccine vehicle in parasitology has never been reported. The EtMic2, a microneme protein of E. tenella, is secreted from the microneme and intimately involved in host-cell invasion [29,30]. Previous studies suggested that the EtMic2 protein showed partial protection of the chicken from E. tenella infections and might be a good candidate for use in vaccine development [11,31,32]. In the present study, we express the EtMic2 on the surface of the S. cerevisiae strain HAO and evaluated it as a potential oral vaccine for chicken against E. tenella infection. To our knowledge, this is the first report to study the EtMic2 as yeast live oral vaccine for its immunogenic and protective efficacy in a chicken challenge model.

(Guava EasyCyte Mini, USA). The S. cerevisiae strains transformed with plasmid pYSD-EtMIC2 and pYSD were named as HAO/pYSDEtMIC2 and HAO/pYSD, respectively. 2.4. Western blot analysis of EtMic2 The western blot assay was carried out to further test whether the EtMic2 protein was displayed on the yeast surface. Freshly harvested 4 × 107 yeast cells were washed twice with ice-cold PBS, resuspended in PBS supplemented with 100 mM DTT (Sigma, USA), and incubated for 4 h at 4 ◦ C to release the Aga2p-EtMic2 fusion protein from the cell surface. Proteins were separated by 12% SDS-PAGE and transferred to PVDF membrane (Millipore, USA). Aga2p-EtMic2 fusion protein was detected with 1:10 diluted antiEtMic2 monoclonal antibody (prepared in our lab) and horseradish peroxidase-conjugated goat anti-mouse (1:2000, TransGen, China). 2.5. Immunization and parasite-challenge infection

2. Materials and methods

Freshly harvested 1 × 108 yeast cells used for vaccination were washed 3 times in sterile PBS before being resuspended in 1 ml 1× PBS buffer and administered at 1 × 107 /chicken using oral gavage to chickens from 2 day-old to 21 day-old. At 21 days post immunization, all groups were orally challenged with 3000 freshly sporulated E. tenella oocysts except the unchallenged control (group I). The number of challenged E. tenella sporulated oocysts was determined by our pre-tests (unpublished data).

2.1. Experimental chickens

2.6. Body weight gain and fecal oocyst shedding measuring

One day-old male Hy-Line variety brown layer chickens (Dongyue poultry, Taian, China) were reared in clean cages and provided with coccidiostat-free feed and water ad libitum. Chickens were randomly assigned to four groups of 20 birds per group (Table 1).

Body weights of the chickens in each group were measured at 2 d, 21 d (before challenge n = 20) and 28 d (7 d post-challenge n = 17). In brief, the body weight gain was calculated as follows: the body weight at 21 d or 28 d minus the body weight at 2 d or 21 d. The relative body weight gain was calculated as follows: (the average body weight gain of chickens in immunized group or challenged control group)/(the average body weight gain of chickens in the unchallenged control group) × 100% [35]. Fecal oocysts numbers for each group were counted from 1 g of feces using McMaster’s counting technique. The percentage decrease in oocyst shedding was calculated as follows: (the number of oocysts from control birds minus the number of oocysts from vaccinated birds)/the number of oocysts from control birds × 100% [36].

2.2. Coccidial oocysts Wild type E. tenella strain SD-01 was stored in our laboratory and propagated in 2 weeks old chickens every 6 months. Propagation and purification of oocysts followed the method described by Fetterer and Barfield [33] with some modifications. In brief, two week-old chickens were infected with 3000 sporulated E. tenella oocysts and the chicken caeca and their contents were collected on day 7 post-infection. The oocysts were purified by floating with saturated NaCl solution, washed three times with saline, and resuspended in 2% potassium dichromate solution. The oocysts were incubated at 28 ◦ C for 72 h for sporulation. 2.3. Yeast surface display, indirect immunofluorescence assay and flow cytometry analysis of EtMic2 The construction of the EtMic2 displaying vector, yeast transformation, surface display of the EtMic2, indirect immunofluorescence assay, and flow cytometry analysis were performed as described previously [34]. Briefly, the EtMic2 expression plasmid pYSDEtMIC2 and the empty plasmid pYSD (both plasmids were made and stored in our lab) were transformed into S. cerevisiae HAO strain and the correct transformants were screened on the YPD plate containing G418 at 100 ␮g/ml. Then the positive transformants screened out were cultured for displaying EtMic2 protein on the yeast surface. The displayed EtMic2 protein was detected by the immunofluorescent labeling assay using specific anti-EtMic2 primary antibody and FITC-conjugated goat anti mouse secondary antibody. Following fluorescence detection, the yeasts displayed EtMic2 stained with IFA were analyzed using flow cytometer

2.7. Determination of cecal lesion scores At 7 day post challenge, the lesion score of chickens (n = 17) from each group was evaluated using a numerical scale from 0 (normal) to 4 (severe) following the method described by Johnson and Reid [35]. A score 0 = no gross lesions; score 1 = a few scattered white lesions; score 2 = lesions are much closer together but discrete; the intestinal walls show no thickening; score 3 = lesions are extensive enough to cause coalescence; the intestinal walls are thickened and the contents are watery; score 4 = the mucosal walls are grayish with extensive coalescence of lesions, the intestinal walls are thickened and the intestine is filled with bloody contents. The lesion scores were evaluated by three independent observers. 2.8. Serum antibody and cecal sIgA measurement At 21 d (before challenge) and 28 d (7 d post-challenge), three chickens were used for the serum antibody and cecal sIgA measurement. Firstly, the chicken peripheral blood was drawn from the heart and the sera were prepared by low speed centrifugation. Then, chickens were killed by cervical dislocation, caeca were cut longitudinally and content were removed, and the cecal washes

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were collected following the method described by Jang et al. [37]. The sera and the cecal washes were used to measure EtMic2 antibody and sIgA level by ELISA, respectively. Microtiter plates were coated with EtMic2 protein (expressed in E. coli made in our lab, 200 ng/well) overnight at 4 ◦ C, then the plates were washed with PBS containing 0.05% Tween-20 (PBST), and blocked with 5% nonfat milk in PBST for 1 h at 37 ◦ C. After washing with PBST, 100 ␮l diluted serum samples (1:10) or cacel washes were added into each well and incubated for 1 h at 37 ◦ C. The plates were washed and 100 ␮l of the second antibody (horseradish peroxidase-conjugated rabbit anti-chicken IgY or IgA, purchased from Bethyl, USA) with 1:1000 dilution was added and incubated for 1 h at 37 ◦ C. Optical density values at 450 nm (OD450) were measured using an automated microplate reader (Biotek, USA). All samples were analyzed in triplicate. 2.9. Blood lymphocyte proliferation assay (LPA) Blood lymphocytes were isolated using the lymphocyte separation medium (Solarbio, China) following the manufacturer’s instruction. The lymphocytes were washed three times with RPMI 1640 supplemented with 10% fetal bovine serum (Gibco, USA) and their viability was estimated by trypan blue dye exclusion. The lymphocytes were resuspended in culture medium (RPMI 1640 supplemented with 0.01% antibiotics and 10% fetal bovine serum) at a concentration of 1 × 107 cells/ml. Lymphocytes (1 × 106 cells) and 10 ␮l of ConA (Solarbio, China, 0.5 mg/ml) were added into each well of microtiter plates and cultured at 37 ◦ C in 5% CO2 supplement. At the 66 h of cultivation, 10 ␮l of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) solution (Solarbio, China, 5 mg/ml) was added to each well and continue to incubate for 4 h. Then each well was added with 100 ␮l of a dimethylsulphoxide (DMSO) and plates were kept on orbital shaker for 5 min and OD570 was read on ELISA reader after 10 min. All samples were analyzed in triplicate. 2.10. Analysis of chicken cytokines Sera IFN-␥ and IL-18 at 28 days (7 d post-challenge) were quantified using chicken IFN-␥ or IL-18 ELISA kit (Elabscience Biotechnology, China) according to the manufacturer’s instruction. All samples were analyzed in triplicate. 2.11. Statistical analysis All data were analyzed by one-way analysis of variance (ANOVA) followed by the Duncan’s multiple range using SPSS 16.0 for Windows (SPSS Inc., Chicago, IL). Differences between groups were considered statistically significant at P < 0.05.

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surface was analyzed using flow cytometry. About 70% HAO cells transformed pYSD-MIC2 were display EtMic2 on their surface (Fig. 1C). The HAO/pYSD strain was used as the negative control (Fig. 1F). Since the antibodies cannot enter the yeast cell, the results confirm that EtMic2 is displayed on the surface of the yeast cell. 3.2. Expression of EtMic2 detected by western blot The western blot analysis was employed to further demonstrate that the EtMic2 was displayed on the yeast cell surface. Because the Aga2-EtMic2 fusion protein is linked to the cell wall anchor protein Aga1p through two S S bonds, the Aga2pEtMic2 fusion protein can be released by DTT treatment. The DTT extracted Aga2p-EtMic2 fusion protein was detected using antiEtMic2 monoclonal antibody. The Aga2p-EtMic2 fusion protein is predicted to encode polypeptide of 489 amino acids with a deduced molecular mass of 54.8 KD. The results showed that DTT extractions from the HAO/pYSD-EtMIC2 strain exhibited the specific bands at about 55–60 kD positions which are similar to the predicted size. Whereas no band was detected in HAO/pYSD control (Fig. 2). 3.3. Evaluation of the protective effect of HAO/pYSD-EtMIC2 3.3.1. Body weight gain The efficacy of immunization was evaluated on the basis of body weight gain, lesion score and oocyst shedding. No death occurred in all experimental groups. Chickens in groups III and IV were immunized with HAO/pYSD and HAO/pYSD-EtMIC2, respectively, and then challenged with 3000 E. tenella oocysts. Weight gains were assessed at days 2, 21 (before challenge) and 28 (7 d post-challenge). At 21 d (before challenge), chickens in group III and IV had 106% and 105% increase on weight gain compared to control group I (Table 2). At 28 d (7 d post-challenge), the relative body weight gain in group IV was 80.9% compared to control group I, which is significantly higher than that in both mock immunizedchallenged chickens (64.5%) and non-immunized-challenged chickens (60.3%, Table 2). 3.3.2. Cecal lesion scores The cecal lesion scores were measured according to the method described by Johnson and Reid [35] using a numerical scale from 0 (normal) to 4 (severe). The average cecal lesion scores in unimmunized controls (group II), controls immunized with HAO/pYSD (group III), and chickens immunized with HAO/pYSDEtMIC2 (group IV) were recorded as +4.0, 3.6 ± 0.51, and 2.2 ± 0.44 respectively (Table 2). The cecal lesion score of chickens immunized with HAO/pYSD-EtMIC2 was significantly decreased than that of the both control groups.

3. Results 3.1. Displaying the EtMic2 on the yeast surface To make the live oral vaccine of E. tenella using S. cerevisiae as vehicle, the plasmid pYSD-EtMIC2 was transformed into S. cerevisiae HAO strain to obtain the HAO/pYSD-EtMIC2 strain that expressed EtMic2 protein on the yeast surface. The plasmid pYSD was also transformed to make the HAO/pYSD strain used as control. The HAO/pYSD-EtMIC2 strain and the HAO/pYSD strain were detected by the immunofluorescent labeling assay using specific anti-EtMic2 primary antibody and FITC-conjugated goat anti mouse secondary antibody. The results showed that the green fluorescence was homogeneously distributed on the HAO/pYSD-EtMIC2 surface (Fig. 1B) and there was no fluorescence on the HAO/pYSD strain (Fig. 1E). Quantitative expression of EtMic2 on yeast cell

3.3.3. Fecal oocysts shedding The oocysts shedding of each group was measured and the percentage decrease in fecal oocyst shedding was calculated. Compared with the unimmunized-challenged controls (group II), the oocyst shedding of chickens immunized with HAO/pYSD-EtMIC2 (group IV) was reduced 76.7% and that of chickens immunized with HAO/pYSD (group III) was reduced 62.5% (Table 2). The protective effect of HAO/pYSD-EtMIC2 evaluated on the basis of body weight gain, lesion score and oocyst shedding was consistent with the pre-experimental data. In the pre-experiment, one day-old male Hy-Line variety brown layer chickens were randomly assigned to five groups of 25 birds per group. In addition to four groups as described in Table 1, the fifth group is immunized with S. cerevisiae HAO (107 /chicken). All chickens were used to detect the relative body weight gain, lesion score and oocyst

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Fig. 1. Microscopic and flow cytometric photographs of HAO/pYSD-MIC2 and HAO/pYSD. (A) HAO/pYSD-MIC2 under normal white light. (B) HAO/pYSD-MIC2 under the FITC filter. (D) HAO/pYSD under normal white light. (E) HAO/pYSD under the FITC filter. Flow cytometric dot plots shown in C and F depict the mean fluorescent signal of EtMic2 displayed on yeast cells (in red) and control cells (in blue). The scale bars in panels A–B, D–E represent 10 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

shedding according to the methods described in Sections 2.6 and 2.7. The results showed that there were no significant differences between the group V (immunized with HAO) and the group III (immunized with HAO/pYSD) in the body weight gain, cecal pathology and fecal oocyst shedding (Table 3). After infection, the relative body weight gain in group IV was 81.1% compared to control group I, the oocyst shedding of chickens was reduced 74.0% and the

average lesion score was 2.3 ± 0.46. The pre-experimental data was summarized in Table 3. 3.4. Serum antibody and cecal sIgA levels In order to evaluate the humoral response, the sera and the cecal washes were tested for anti-EtMic2 specific antibody and sIgA

Table 2 Protective effects of the HAO/pYSD-EtMIC2 against E. tenella in chicken. Group

Average body weight gain (g)

21 d I II III IV

117.94 115.48 125.02 123.50

Relative body weight gain (%)

28 d ± ± ± ±

b

11.78 11.15b 12.87a 13.81a

63.15 38.08 40.73 51.09

± ± ± ±

a

14.56 10.11d 15.83c 12.92b

21 d

28 d

100 97.91 106.00 104.71

100 60.30 64.49 80.90

Values with different letters in the same column are significantly different (P < 0.05).

Oocyst shedding (×106 )

Decrease in oocyst shedding (%)

Lesion score

0 1.28 ± 0.04a 0.48 ± 0.03b 0.30 ± 0.02c

100 0 62.50 76.56

0 +4.0a 3.6 ± 0.51a 2.2 ± 0.44b

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Table 3 Pre-experimental data showing protective effects of the HAO/pYSD-EtMIC2 against E. tenella in chicken. Group

Average body weight gain (g)

21 d I II III IV V

124.56 123.01 132.13 130.28 133.43

Relative body weight gain (%)

28 d ± ± ± ± ±

c

15.01 14.13c 15.19ab 13.06b 12.63a

59.67 35.83 37.69 48.38 38.51

± ± ± ± ±

a

11.38 12.41d 13.27c 13.18b 14.03cd

21 d

28 d

100 98.76 106.07 104.59 107.12

100 60.47 63.16 81.08 64.54

Oocyst shedding (×106 )

Decrease in oocyst shedding (%)

Lesion score

0 1.19 ± 0.04a 0.41 ± 0.02b 0.23 ± 0.02c 0.44 ± 0.03b

100 0 65.54 73.95 63.03

0 +4.0a 3.7 ± 0.46b 2.3 ± 0.46c 3.9 ± 0.3ab

Values with different letters in the same column are significantly different (P < 0.05).

Fig. 2. Western blot analysis of Aga2p-EtMic2 fusion protein. The HAO/pYSDEtMIC2 (lane A) and HAO/pYSD (lane B) was treated by DTT and detected Aga2p-EtMic2 fusion protein using anti-EtMic2 monoclonal antibody. The expected Aga2p-EtMic2 fusion protein is about 55 KD.

using ELISA. Both the serum anti-EtMic2 antibody and the cecal sIgA were increased in group IV and showed significantly higher than that in group III at 21 days and 28 days. The serum antiEtMic2 antibody level was significantly higher at 28 days than 21 d in HAO/pYSD-EtMIC2 group (Fig. 3A). At the two time points, there was no significantly difference between HAO/pYSD group and the unimmunized and challenged group in both anti-EtMic2 antibody and cecal sIgA levels (Fig. 3).

Fig. 3. Effects of immunized with HAO/pYSD-EtMIC2 on EtMic2 and sIgA antibody levels. Chickens were orally immunized with strain HAO/pYSD or strain HAO/pYSDEtMIC2 and challenge with 3000 E. tenella oocysts (except for unchallenged control). EtMic2 levels (A) and sIgA (B) were measured by ELISA at 21 d (before challenge) and 28 d (7 d post-challenge). Each bar represents the mean ± SD values (n = 3) and each sample was analyzed in triplicate. Bars not sharing the same letters were significantly different according to the Duncan’s multiple range (P < 0.05).

3.5. Proliferation of blood lymphocytes As an indicator of cell-mediated immune, the ability of lymphocytes to proliferate in vitro in response to mitogen was measured by MTT. The lymphocyte proliferation response to ConA was significantly higher in HAO/pYSD-EtMIC2 immunized chickens (group IV) than that in the HAO/pYSD immunized chickens (group III) at 7 d post-challenge (Fig. 4). There was no significant difference among three groups (HAO/pYSD group, the unchallenged group and the unimmunized and challenged group).

3.6. Evaluation of IFN- and IL-18 expression level in sera The ELISA results of IFN-␥ and IL-18 expression level in sera are shown in Fig. 5. The IFN-␥ and IL-18 levels of HAO/pYSD-EtMIC2 group are higher than HAO/pYSD group at 28 d, and there was no significant difference between HAO/pYSD group and the unimmunized and challenged group (Fig. 5).

Fig. 4. Effects of immunized with HAO/pYSD-EtMIC2 on proliferation of blood lymphocyte. Chickens were orally immunized with strain HAO/pYSD or strain HAO/pYSD-EtMIC2 and challenge with 3000 E. tenella oocysts (except for unchallenged control). The ability of lymphocytes to proliferate in vitro in response to ConA was measured by MTT at 28 d (7 d post-challenge). Each bar represents the mean ± SD values (n = 3) and each sample was analyzed in triplicate. Bars not sharing the same letters were significantly different according to the Duncan’s multiple range (P < 0.05).

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Fig. 5. Effects of immunized with HAO/pYSD-EtMIC2 on cytokines IFN-␥ and IL-18. Chickens were orally immunized with strain HAO/pYSD or strain HAO/pYSD-EtMIC2 and challenge with 3000 E. tenella oocysts (except for unchallenged control). The IFN-␥ (A) and IL-18 (B) level was measured by ELISA at 28 d (7 d post-challenge). Each bar represents the mean ± SD values (n = 3) and each sample was analyzed in triplicate. Bars not sharing the same letters were significantly different according to the Duncan’s multiple range (P < 0.05).

4. Discussions Chicken coccidiosis causes tremendous economic losses and environmental issues [1,2]. As the emergence of drug resistance of Eimeria species and the environmental pollution of the live oocyst vaccine [3,6], the development of new vaccine types against Eimeria species infections has draw more attentions of parasitologists worldwide [6,7]. In the present study, we expressed the EtMic2 protein of E. tenella on the cell surface of the probiotic yeast S. cerevisiae and used the whole yeast cells with EtMic2 protein expressed on their surfaces as live oral vaccine to immunize chickens against E. tenella challenge. The effect of the immunization was mainly evaluated by the cecal lesion score, oocyst shedding, and weight gain. The antibody levels in the sera and the cecal mucous membrane, and the proliferation ability of blood lymphocytes were also considered in the evaluation. The results showed that the oocyst shedding and the lesion scores were significantly lower (P < 0.05) than control groups (the unimmunized and challenged group and the unimmunized with yeast without EtMic2 protein expressed group), while the weight gains of vaccinated groups were significantly increased (P < 0.05) than that of control chickens. The cecal lesion score, the oocyst shedding, and the weight gain are the main criteria of the disease burden of chicken coccidiosis. Reducing the disease burden has some practical significant in poultry industry and it is regarded as the evaluation criteria for the evaluation of vaccine efficacy and protective immunity in avian coccidiosis [38]. Our results revealed that the yeast live oral vaccine we developed in the present study can offer partial protection against E. tenelle infection. Numerous studies have been carried out worldwide to elucidate the mechanism of protective immunity against coccidiosis. It was believed from early studies that cellular immunity plays the most important role in protection against Eimeria spp., whereas humoral immunity plays a very minor role in resistance against infection [32,39]. However, recent results showed the ability of antibodies (raised by live immunization or against purified

stage-specific Eimeria antigens) to inhibit parasite development in vitro and in vivo and demonstrated the role of antibody in protection against coccidiosis [40,41]. To evaluate the effect of our new live oral vaccine, we also measured the antibody levels, the proliferation ability of blood lymphocytes, and IFN-␥ and IL-18 levels. The results revealed that anti-EtMic2 protein specific antibodies in blood and cecal mucous membrane were significantly higher (P < 0.05) in the live oral vaccine immunized group than in mockimmune group, and the proliferation ability of blood lymphocytes is significant higher in HAO/pYSD-EtMIC2 immunized group than in mock immunized group, which suggests that the oral live vaccine can stimulate the host to develop both humoral and cellular immunity against E. tenella infection. IFN-␥ and IL-18 are Th1 or Th1 related cytokines produced by mononuclear phagocytes and other cell in response to intracellular pathogen infections, such as viral or protozoan infection [42–44]. The expression levels of IFN-␥ and IL-18 were significantly higher in chickens vaccinated with HAO/pYSD-EtMIC2 compared with mockimmunized control. IFN-␥ can inhibit the intracellular development of E. tenella when injected the recombinant chicken IFN-␥ [45]. Dalloul et al. reported transcript levels of IL-18 highly up-regulated during experimental coccidiosis [46]. The high expression level of cytokine may enhance the HAO/pYSD-EtMIC2 protection against coccidiosis by promoting adaptive immunity. Several prospective studies on developing live oral vaccines against different kinds of pathogens using virus, bacteria, and yeasts as antigen delivery vehicles [21,47–49] were reported. For parasitic pathogens, yeast platform has some specific advantages over other antigen deliver systems. It is not only because the yeast is a eukaryotic organism that is similar to higher eukaryotes in post-translational processing, but also because the S. cerevisiae is probiotic yeast that can promote the host’s innate immunity and improve the productivity of the host, greatly increase the vaccine efficiency. Several studies reported that the S. cerevisiae fermentation product could promote the growth rate and immune function of the chicken with the E. tenella infection [50–52]. Actually, S. cerevisiae has long been used in poultry industry to improve body weight gain [50,53,54]. Our results in the present study are consistent with the previous studies. Weight gains in both group III and group IV at days 21 (before challenge) and 28 (7 d post-challenge) were significantly higher than that in control group I. The oocyst shedding in the group III was also decreased significantly than that in the unimmunized and challenged group (group II). The average cecal lesion score in group III (3.6 ± 0.51) showed lower than that in the unimmunized controls (group II, 4.0) although decrease is not significant. The results suggest that S. cerevisiae itself provide some protections against E. tenella infection. Du and Wang [12] evaluated the efficacy of the oral vaccine using an attenuated Salmonella typhimurium as vehicle carrying the E. tenella 5401 antigen. Although the vaccine could provide some protection against E. tenella infection, there is no any improvement in weight gain between the vaccinated group and the control group before challenge. Zhang et al. [55] evaluated the protection effect against E. tenella challenge using EtMic2 recombinant sub-unit antigen vaccine. Their results showed that the percentage of oocyst shedding and the lesion scores are significantly lower than the unimmunized and challenged group. The oocyst shedding in the group immunized with EtMic2 recombinant sub-unit vaccine decreased 15.7% compared with controls [55]. In the present study, the oocyst shedding in the group immunized with EtMic2 yeast live oral vaccine decreased 76.7% compared with controls. Our results showed that EtMic2 yeast live oral vaccine offered better protection in deducing oocyst shedding for chickens against E. tenella infection. Probably the biggest advantage of the yeast live oral vaccine is its simple to use, just as Hippocrates said [13], “Let thy food

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be thy medicine”. The feed additive, S. cerevisiae, has a positive impact on the animal health through its direct nutritional effect when administered through the digestive tract [56]. The EtMic2 yeast oral vaccine as the feed additive has high potential to fight against coccidiosis and it is adapt to using it in large scale. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements 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). References [1] Williams RB. Anticoccidial vaccines for broiler chickens: pathways to success. Avian Pathol 2002;31(4):317–53. [2] Regulation (EC) No.1831/2003 of the European Parliament and of the council of 22 September 2003 on additives for use in animal nutrition. Off J Eur Union 2003;L268:29–43. [3] Liu Y, Zheng J, Li J, Gong P, Zhang X. Protective immunity induced by a DNA vaccine encoding Eimeria tenella rhomboid against homologous challenge. Parasitol Res 2013;112:251–7. [4] Vermeulen AN, Schaap DC, Schetters TP. Control of coccidiosis in chickens by vaccination. Vet Parasitol 2001;100(1–2):13–20. [5] Williams RB. Fifty years of anticoccidial vaccines for poultry (1952–2002). Avian Dis 2002;46:775–802. [6] Vermeulen AN. Progress in recombinant vaccine development against coccidiosis. A review and prospects into the next millennium. Int J Parasitol 1998;28(7):1121–30. [7] Lillehoj HS, Lillehoj EP. Avian coccidiosis: a review of acquired intestinal immunity and vaccination strategies. Avian Dis 2000;44:408–25. [8] Shams H. Recent developments in veterinary vaccinology. Vet J 2005;170(3):289–99. [9] Shin MK, Kang ML, Jung MH, Cha SB, Lee WJ, Kim JM, et al. Induction of protective immune responses against challenge of Actinobacillus pleuropneumoniae by oral administration with Saccharomyces cerevisiae expressing Apx toxins in pigs. Vet Immunol Immunopathol 2013;151(January (1/2)):132–9. [10] Min W, Lillehoj HS, Burnside J, Weining KC, Staeheli P, Zhu JJ. Adjuvant effects of IL-1beta, IL-2, IL-8, IL-15, IFN-alpha, IFN gamma TGF-beta4 and lymphotactin on DNA vaccination against Eimeria acervulina. Vaccine 2001;20:267–74. [11] Ding X, Lillehoj HS, Dalloul RA, Min W, Sato T, Yasuda A. In ovo vaccination with the Eimeria tenella EtMIC2 gene induces protective immunity against coccidiosis. Vaccine 2005;23(28):3733–40. [12] Du A, Wang S. Efficacy of a DNA vaccine delivered in attenuated Salmonella typhimurium against Eimeria tenella infection in chickens. Int J Parasitol 2005;35(7):777–85. [13] Jacob SS, Cherian S, Sumithra TG, Raina OK, Sankar M. Edible vaccines against veterinary parasitic diseases – current status and future prospects. Vaccine 2013;31(15):1879–85. [14] Chaudhari AA, Matsuda K, Lee JH. Construction of an attenuated Salmonella delivery system harboring genes encoding various virulence factors of avian pathogenic Escherichia coli and its potential as a candidate vaccine for chicken colibacillosis. Avian Dis 2013;57(1):88–96. [15] Qiu L, Wang X, Hao H, Mu G, Dang R, Wang J, et al. Oral administration of attenuated Salmonella typhimurium containing a DNA vaccine against rabbit haemorrhagic disease. J Virol Methods 2013;188(1–2):108–13. [16] Phalipon A, Sansonetti P. Live attenuated Shigella flexneri mutants as vaccine candidates against shigellosis and vectors for antigen delivery. Biologicals 1995;23(2):125–34. [17] Yang XF, Qu XZ, Wang K, Zheng J, Si LS, Dong XP, et al. Construction of prophylactic human papillomavirus type 16 L1 capsid protein vaccine delivered by live attenuated Shigella flexneri strain sh42. Acta Biochim Biophys Sin (Shanghai) 2005;37(11):743–50. [18] Zheng JP, Zhang ZS, Li SQ, Liu XX, Yuan SL, Wang P, et al. Construction of a novel Shigella live-vector strain co-expressing CS3 and LTB/STm of enterotoxigenic E. coli. World J Gastroenterol 2005;11(22):3411–8. [19] Soussi N, Saklani-Jusforgues H, Colle JH, Milon G, Glaichenhaus N, Goossens PL. Effect of intragastric and intraperitoneal immunisation with attenuated and wild-type LACK-expressing Listeria monocytogenes on control of murine Leishmania major infection. Vaccine 2002;20(21–22):2702–12. [20] Muller WJ, Orgun NN, Dong L, Koelle DM, Huang ML, Way SS. Recombinant Listeria monocytogenes expressing an immunodominant peptide fails to protect after intravaginal challenge with herpes simplex virus-2. Arch Virol 2008;153(6):1165–9.

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