Experimental Parasitology 141 (2014) 75–81

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Immunization with recombinant 3-1E protein in AbISCOÒ-300 adjuvant induced protective immunity against Eimeria acervulina infection in chickens Ying Zhang a,1, Lixia Wang b,1, Wenke Ruan a, Jianjun Zhang a, Peng Yao a, Sai Zhou a, Jian An a,⇑ a b

College of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206, People’s Republic of China School of Animal Science and Veterinary Medicine, Beijing Vocational College of Agriculture, Beijing 102442, People’s Republic of China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 The soluble recombinant protein of

E. acervulina 3-1E gene was expressed in E. coil.  Immunization with protein plus adjuvant increased intestinal CD3+, CD4+, CD8+cells.  Immunization with protein plus adjuvant increased positive expression rate of IgA.  Immunization with protein plus adjuvant increased post-infection body weight gain.  Immunization with protein plus adjuvant decreased fecal oocyst shedding.

a r t i c l e

i n f o

Article history: Received 25 December 2013 Received in revised form 27 February 2014 Accepted 4 March 2014 Available online 24 March 2014 Keywords: Eimeria acervulina Recombinant 3-1E protein Adjuvant Chicken Coccidiosis

a b s t r a c t Immunostimulating complexes (ISCOMs), a kind of novel antigen presenting system, could enhance immune protection by antigen presentation. AbISCOÒ-300 comprising purified saponin, cholesterol and phosphatidyl choline is an effective ISCOM adjuvant. To evaluate the immune protection of recombinant 3-1E protein against Eimeria acervulina infection, chickens were immunized with recombinant 3-1E protein in combination with AbISCOÒ-300 or recombinant 3-1E protein alone in this study. The protective immunity was assessed with body weight gain, fecal oocyst output, detection of intestinal IgA positive cells and percentages of CD3+, CD4+ or CD8+ intestinal intraepithelial lymphocytes (IELs). Chickens vaccinated with different doses of recombinant 3-1E protein plus AbISCOÒ-300 showed higher percentages of CD3+, CD4+, and CD8+ intestinal IELs, increased positive expression rate of intestinal IgA, increased body weight gains and decreased oocyst shedding compared with recombinant 3-1E protein-only vaccinated groups. The results showed that immunization with various doses of the recombinant 3-1E protein in AbISCOÒ-300 adjuvant enhanced immune protection against avian coccidiosis. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction

⇑ Corresponding author. 1

E-mail address: [email protected] (J. An). Ying Zhang and Lixia Wang contributed equally to this work.

http://dx.doi.org/10.1016/j.exppara.2014.03.001 0014-4894/Ó 2014 Elsevier Inc. All rights reserved.

Avian coccidiosis caused by Eimeria spp. infection results in great losses globally in the poultry industry every year (Lillehoj et al., 2000). Eimeria acervulina (E. acervulina) is one of the most

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Y. Zhang et al. / Experimental Parasitology 141 (2014) 75–81

prevalent Eimeria species in chicken. E. acervulina parasitized mainly in the duodenum. Infectious parasites invade intestinal epithelial cells, eliciting a variety of clinical manifestations including necrotic gut lesions, inefficient feed utilization, impaired growth rate, and, in severe cases, mortality (Min et al., 2013). Conventional coccidiosis control relies primarily on prophylactic chemotherapy, but drug resistance and drug residues in meats or meat products render chemotherapy an unsustainable control method against coccidiosis. Efforts have been made to the development of alternative control strategies against coccidiosis. One of the most effective methods in veterinary practice is the vaccination with controlled live coccidia (Williams et al., 1999; Williams and Catchpole, 2000). However, giving chicken attenuated vaccine might cause coccidiosis outbreaks. Therefore, researchers are looking for safer and more effective vaccines against avian coccidiosis through the induction of protective innate and adaptive immunity by vaccination with one or more antigens in combination with an adjuvant. 3-1E is the common surface antigen of E. acervulina sporozoites and merozoites and it is expressed at both stages of sporozoites and merozoites (Lillehoj et al., 2000). DNA vaccine immunization of 3-1E gene can induce partial immune protection against coccidiosis (Lillehoj et al., 2000; Ma et al., 2011; Min et al., 2001; Song et al., 2000). It is suggested that 3-1E protein is a protective antigen and worth further investigation. ISCOMs which are novel antigen presenting adjuvants could enhance immune protection by antigen presentation. Previous studies showed the effects of ISCOM adjuvant on the immunity against chicken coccidiosis (Berezin et al., 2008, 2010). The antigens they used were crude antigens prepared by detergent extraction mixture of oocysts, sporozoites and merozoites, which contain multiple proteins and may have other unknown immunologic functions. In this experiment, recombinant 3-1E protein was used as antigen to investigate the immune protection of the vaccine against E. acervulina infection. 2. Materials and methods 2.1. Parasites Beijing strain E. acervulina was maintained by passage every 3 months in susceptible chickens at the College of Animal Science and Technology, Beijing University of Agriculture, China. 2.2. Chickens One-day-old male layer chickens were obtained from a hatchery (Changping, Beijing), provided with feed and water ad libitum. Chickens were kept in brooder pens in a coccidia-free facility for 21 days post-hatch and transferred into large cages at a separate location where they were infected and kept until the end of the experiment. 2.3. Cloning, expression, and purification of E. acervulina recombinant 3-1E protein E. acervulina recombinant 3-1E protein was expressed in Escherichia coli as described (Yao et al., 2012). Briefly, E. acervulina 3-1E gene was cloned by PCR into the pET28a vector with a His tag. The constructed vector was identified by double enzyme digestion and DNA sequence analysis, and then transformed into competent E. coli cells. The bacteria were cultured for 4 h at 37 °C, and induced for 50 h at 16 °C with 0.4 mM isopropyl b-D-1-thiogalactopyranoside. Bacteria were harvested by centrifugation at 5000 rpm for 10 min at 4 °C, resuspended in bacteria protein extraction reagent

(CWBIO, China), incubated for 40 min at 37 °C, and centrifuged at 12,000 rpm for 20 min at 4 °C. The supernatant was purified using the His-tagged protein purification kit (CWBIO, China). Protein purity was confirmed on Coomassie blue-stained SDS–polyacrylamide gels. The purified protein was stored at 80 °C until use. 2.4. Vaccine formulation The concentration of recombinant 3-1E protein was determined with BCA protein quantitation kit (Aidlab, China) according to the instruction. AbISCOÒ-300 (Isconova AB, Sweden) is a liquid reagent and its concentration is 2.7 mg/mL (50 lg/bird). The recombinant 3-1E protein and adjuvant were mixed after calculating their volumes according to the number of chickens. The final volume was 200 lL/bird using PBS to supplement. 2.5. Experimental design Chickens were randomly divided into 8 treatment groups (Table 1): (1) 3-1E (1 lg/bird) plus AbISCOÒ-300, (2) 3-1E (5 lg/ bird) plus AbISCOÒ-300, (3) 3-1E (10 lg/bird) plus AbISCOÒ-300, (4) 3-1E (1 lg/bird), (5) 3-1E (5 lg/bird), (6) 3-1E (10 lg/bird), (7) unimmunized and infected controls, (8) unimmunized and uninfected controls. Control groups were immunized with PBS. At 1 week of age, chickens were intramuscularly immunized. At 1 week post primary immunization, chickens were given an identical secondary immunization. At 7 days post-secondary immunization, chickens were orally challenged with 1  105 sporulated E. acervulina oocysts. The unimmunized and uninfected controls were orally challenged with PBS (Fig. 1). 2.6. Flow cytometry At 14, 21 and 28 day old, 6 birds from each group were euthanized by cervical dislocation before duodena were collected. Duodenum intestinal intraepithelial lymphocytes (IELs) were isolated according to the previously described method (Hong et al., 2006; Zhang et al., 2012). Briefly, duodena were cut longitudinally and washed five times with ice-cold Hank’s balanced salt solution (HBSS) containing 100 U/mL of penicillin and 100 mg/mL of streptomycin (M&C gene Technology, China). The tissue was incubated in HBSS containing 0.5 mM EDTA and 5% fetal calf serum for 20 min at 37 °C with constant swirling. Released cells were further purified using a commercial lymphocyte separation medium (M&C gene Technology, China) with centrifugation at 2000 rpm for 20 min at room temperature. IELs were resuspended in HBSS, incubated with mouse anti-chicken CD3+, CD4+ and CD8+ antibodies (Southern Biotech, USA), and analyzed by flow cytometry (Beckman Coulter) as described (Min et al., 2001). 2.7. Immunohistochemistry At 14, 21 and 28 day old, duodenum segments were excised and fixed in 4% paraformaldehyde for 48 h and embedded in paraffin. After deparaffinizing, the sections were heated in microwave oven (in 0.1 M citrate buffer pH 6.0, for 20 min) for epitope retrieval. Endogenous peroxidase activity was inactivated by incubation with 3% hydrogen peroxide. Immunostaining was performed by incubating tissue sections with Anti-Chicken IgA (alpha chain)biotin conjugate antibody (Alpha Diagnoestic International, USA, dilution 1:1000) overnight at 4 °C. Then the tissue sections were washed three times with PBS and incubated with StreptavidinPeroxidase (HRP) conjugate (Alpha Diagnoestic International, USA, dilution 1:500) for 30 min at 37 °C. Staining was finally detected using substrate 3,30 -diaminobenzidine (Bethyl, USA). For control staining, primary antibody was omitted. The positive

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Y. Zhang et al. / Experimental Parasitology 141 (2014) 75–81 Table 1 Summary of the experimental design. Treatment group

Number of birds

Protein

Adjuvant (50 lg/bird)

Primary immunization

Second immunization

Challenge infection

1 2 3 4 5 6 7 8

24 24 24 24 24 24 24 24

1 lg/bird 5 lg/bird 10 lg/bird 1 lg/bird 5 lg/bird 10 lg/bird – –

+ + +     

+ + + + + +  

+ + + + + +  

+ + + + + + + 

Chickens immunized with recombinant 3-1E protein plus AbISCOÒ-300 had significantly decreased fecal oocyst shedding compared with birds vaccinated with 3-1E recombinant protein alone (P < 0.05; Fig. 2B). 3.2. Detection of CD3+, CD4+, and CD8+ IEL subpopulations by flow cytometry

Fig. 1. Schematic outline of the experimental design.

expression rate of intestinal IgA was calculated by using the MIAS medical image analysis software. 2.8. Body weight gain and fecal oocyst shedding Uninfected and Eimeria-infected birds (6/group) were assessed for body weight gain between 0 (21 day-old) and 9 (30 day-old) days post-infection. Fresh fecal samples were collected from day 6 to 9 post-infection. Feces were stored at 4 °C and examined for oocysts daily using a modified McMaster technique (Ding et al., 2004). The results were expressed as number of oocysts per gram of feces (OPG). 2.9. Statistical analysis All data were subjected to one-way analysis of variance (ANOVA) using SPSS 17.0 for Windows (SPSS Inc., Chicago, IL). Mean values of treatment groups were compared using the Tukey test and differences were considered statistically significant at P < 0.05. 3. Results 3.1. Protective effects of the immunization against E. acervulina challenge Efficacy of immunization was evaluated on the basis of the survival rate, body weight gain (BWG), and oocyst shedding (Xu et al., 2013). No death occurred in all challenged groups. The efficacy of the vaccine in terms of body weight gain and OPG was shown in Fig. 2. Uninfected and Eimeria-infected birds were assessed for body weight gain between 0 (21 day-old) and 9 (30 day-old) days post-infection. Body weight gains of recombinant 3-1E protein plus AbISCOÒ-300-vaccinated chickens were greater than those of recombinant 3-1E protein-vaccinated chickens. The significant difference between only 3-1E and 3-1E plus the adjuvant vaccinated groups appeared when 3-1E dose is equal to 5 lg (Fig. 2A).

At 7 days post-primary vaccination, at 7 days post-secondary vaccination and at 7 days post-infection, the percentages of CD3+, CD4+, and CD8+ IEL subpopulations were measured by flow cytometry. Immunization with three different doses of recombinant 3-1E protein plus AbISCOÒ-300 increased intestinal CD3+ IELs at three time points compared with the 3–1E-only group (P < 0.05 in three different doses group at 14-day-old, Fig. 3A; P < 0.05 in 1 lg and 5 lg group at 21-day-old, Fig. 3B; P < 0.05 in 5 lg group at 28-day-old, Fig. 3C). Immunization with 10 lg recombinant 3-1E protein plus AbISCOÒ-300 at 7 days post-secondary vaccination (21-day-old) and immunization with 5 lg recombinant 3-1E protein plus AbISCOÒ300 at 7 days post-infection (28-day-old) increased intestinal CD4+ IELs compared with the 3-1E-only group (Fig. 4B and C). Chickens vaccinated with 1 and 10 lg recombinant 3-1E protein plus AbISCOÒ-300 increased intestinal CD8+ IELs statistically at 14-day-old (P < 0.05, Fig. 5A). Immunization with 1 lg recombinant 3-1E protein plus AbISCOÒ-300 at 7 days post-secondary vaccination (21-day-old) and immunization with 5 lg recombinant 3-1E protein plus AbISCOÒ-300 at 7 days post-infection (28-day-old) increased intestinal CD8+ IELs compared with the 3-1E-only group (P < 0.05, Fig. 5B and C). 3.3. Detection of intestinal IgA+ cells by immunohistochemistry At 7 days post-primary vaccination, 7 days post-secondary vaccination and at 7 days post-infection, the duodenum samples were collected from all groups to observe the intestinal IgA+ cells by immunohistochemistry and evaluate its positive expression level. It is observed that IgA+ cells were mostly in the roots of the duodenal villi and near the intestinal glands. At three different time points, the number of the IgA+ cells in groups vaccinated with recombinant 3-1E protein plus AbISCOÒ-300 was more than that in groups vaccinated with protein alone. Furthermore, IgA+ cells in the three different doses adjuvant groups increased gradually over time (Fig. 6). The positive expression rate of intestinal IgA increased in chickens vaccinated with recombinant 3-1E protein (5 lg/bird) plus AbISCOÒ-300 at 7 days post-primary vaccination (14-day-old), significantly (P < 0.05, Table 2). At 7 days post-secondary vaccination (21-day-old), immunization with three different doses of recombinant 3-1E protein plus AbISCOÒ-300 increased the positive expression rate of intestinal IgA compared with the 3-1E-only groups (P < 0.05, Table 2). At 7 days post-infection (28-day-old),

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Fig. 2. (A) Effects of vaccination with recombinant 3-1E protein plus AbISCOÒ-300 on body weight gain. At 7 days post-secondary vaccination, the chickens were uninfected or infected with 1.0  105 E. acervulina oocysts and body weight was measured between 0 and 9 days post-infection. (B) Effects of vaccination with recombinant 3-1E protein plus AbISCOÒ-300 on fecal oocyst shedding. Fecal oocyst shedding between 6 and 9 days post-infection were determined. Each bar represents the mean ± S.D. value. ⁄P < 0.05 comparing recombinant 3-1E protein plus AbISCOÒ-300 vs. recombinant 3-1E protein alone.

Fig. 3. Effects of vaccination with recombinant 3-1E protein plus AbISCOÒ-300 on intestinal CD3+ IEL subpopulations. Chickens were immunized and infected as described in Fig. 1. The percentages of CD3+ cells were measured by flow cytometry at 7 days post-primary vaccination (A), at 7 days post-secondary vaccination (B) and at 7 days postinfection (C). Each bar represents the mean ± S.D. value (n = 6). ⁄P < 0.05 comparing recombinant 3-1E protein plus AbISCOÒ-300 vs. recombinant 3-1E protein alone.

immunization with recombinant 3-1E protein (10 lg/bird) plus AbISCOÒ-300 increased the positive expression rate of intestinal IgA compared with the 3-1E-only group (P < 0.05, Table 2).

4. Discussion Previous studies demonstrated that recombinant antigen vaccines against the avian coccidiosis only had limited efficacy, could not fully protect coccidial infections (Ding et al., 2004; Lillehoj et al., 2000; Song et al., 2000). The reasons not providing adequate immune protection against coccidiosis in a variety of Eimeria

infection cases are probably because of stage-specificity and species-specificity of the recombinant antigens. Recent studies showed that a weak pathogen antigen in combination with an adjuvant could provide an adequate level of immune stimulation to improve the protective immunity against challenge infection under the experimental condition (Jang et al., 2011). Immunostimulating complexes (ISCOMs) are one of the most successful antigen delivery systems for viral, microbial and parasite antigens (Agrawal et al., 2003; Kensil, 1996; Morein et al., 2004). The ISCOM adjuvant we studied here is AbISCOÒ-300, a trademark for ISCOM adjuvants but similar to ISCOMATRIX regarding its components. The efficacy of the recombinant 3-1E protein

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Fig. 4. Effects of vaccination with recombinant 3-1E protein plus AbISCOÒ-300 on intestinal CD4+ IEL subpopulations. Chickens were immunized and infected as described in Fig. 1. The percentages of CD4+ cells were measured by flow cytometry at 7 days post-primary vaccination (A), at 7 days post-secondary vaccination (B) and at 7 days postinfection (C). Each bar represents the mean ± S.D. value (n = 6). ⁄P < 0.05 comparing recombinant 3-1E protein plus AbISCOÒ-300 vs. recombinant 3-1E protein alone.

Fig. 5. Effects of vaccination with recombinant 3-1E protein plus AbISCOÒ-300 on intestinal CD8+ IEL subpopulations. Chickens were immunized and infected as described in Fig. 1. The percentages of CD8+ cells were measured by flow cytometry at 7 days post-primary vaccination (A), at 7 days post-secondary vaccination (B) and at 7 days postinfection (C). Each bar represents the mean ± S.D. value (n = 6). ⁄P < 0.05 comparing recombinant 3-1E protein plus AbISCOÒ-300 vs. recombinant 3-1E protein alone.

plus AbISCOÒ-300 adjuvant immunoprotection against E. acervulina infection was assessed in this study. It is generally accepted that fecal oocyst shedding and body weight gain are reliable clinical signs for the evaluation of vaccine efficacy and protective immunity in avian coccidiosis (Lillehoj and Lillehoj, 2000). Our study found that chickens immunized with recombinant 3-1E protein plus AbISCOÒ-300 had decreased fecal oocyst shedding and increased body weight gain compared with birds vaccinated with 3-1E recombinant protein alone (Fig. 2A and B).The major finding is that immunization with recombinant 3-1E protein in AbISCOÒ-300 adjuvant was effective in inducing protective response against challenge infection compared to immunization with recombinant protein alone. To research the immune protection of the 3-1E protein against challenge infections, changes of some important IELs and antibody were examined. Lymphocytes are the main immune effector cells, and the changes of their numbers and subsets reflect the immune status. Chicken intestinal IELs, which consists of B cells, T cells, and natural killer cells with CD8+ T lymphocytes as the main effector cells, play a critical role in the generation of a complex immunoregulatory network during Eimeria infection and the ensuing host response (Lillehoj and Trout, 1996; Neutra, 1998). Coccidian sporozoite and merozoite can stimulate activation and proliferation of T lymphocytes and then differentiate into CD4+ and CD8+

T lymphocytes (Gao et al., 2009). The results of the study showed that vaccination with recombinant 3-1E protein plus AbISCOÒ300 increased intestinal CD3+, CD4+, and CD8+ IEL subpopulations (Figs. 3–5). It suggested that the immunization could enhance the cellular immune response of the chicken. At three time points (14-day-old, 21-day-old and 28-day-old), the percentages of CD3+ cells in the recombinant 3-1E protein plus adjuvant groups of different doses were maintained at a high level (Fig. 3A–C). The immunization also activated CD4+ T lymphocytes (primarily T-helper cells). The CD4+ T lymphocytes secrete cytokines such as IFN2c and IL22 to activate macrophages and modulate the immune response (Arstila et al., 1994). At 14-day-old, the percentages of CD4+ cells in the six immunized groups were between 30% and 70%, which were higher than that of the control group (Fig. 4A). At 21 and 28-day-old, the percentages of CD4+ cells in the adjuvant groups were higher than those of the protein-alone groups, while the overall level of CD4+ cells declined (Fig. 5B, C). The percentages of CD4+ cells in the six immunized groups were between 5% and 25%, indicating that the CD4+ cells are activated at the beginning of the immune period, and then gradually declined. For the CD8+ cells, the recombinant 3-1E protein plus adjuvant groups were maintained at a high level at different time points, while the three protein-alone group had no significant difference compared with the control group (Fig. 5A–C). This may be

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Fig. 6. At 14-day-old (A), 21-day-old (B) and 28-day-old (C), immunohistochemical staining was used to detect IgA positive cells. IgA+ cells (brown, arrow heads) in the duodenum were showed in different groups. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 2 Effects of vaccination with recombinant 3-1E protein plus AbISCOÒ-300 or recombinant 3-1E protein alone on the positive expression rate of intestinal IgA at different time points. Dose (lg)

14-Day-old

21-Day-old

28-Day-old

a

1 5 10 1 5 10 1 5 10

3-1E + AbISCOÒ-300 (%)

3-1E (%)

Mean

SD

Mean

SD

2.10a 2.66 1.18 3.12 2.50 3.38 1.55 1.92 3.81

0.59 1.35 0.58 1.12 1.00 1.08 0.56 0.83 1.48

1.24 0.50 0.87 1.26 1.12 1.29 0.61 1.63 1.54

0.93 0.28 0.33 0.54 0.59 0.53 0.29 0.61 0.76

Values are means and SD (n = 10).

P-value

0.120 0.000 0.958 0.000 0.005 0.000 0.096 0.988 0.000

related to low dose of protein. At 28 day old, it is obvious that the percentages of CD3+,CD4+ and CD8+ cells in the 5 lg protein plus adjuvant group were significantly higher than those of 5 lg protein-alone group (Figs. 3C, 4C, 5C), indicating that maybe 5 lg is an appropriate dose. IgA which is a secretory and the characteristic and predominant immunoglobulin of the mucosal immune system plays a key role in immune protection at the level of the mucous membrane surface (Budec et al., 2007; Woof and Kerr, 2006). In order to investigate the specific distribution and changes of the IgA+ cells in the parasitic parts, the duodenum samples were analyzed by immunohistochemistry. It is observed that IgA+ cells were mostly in the roots of the duodenal villi and near the intestinal glands (Fig. 5). The IgA secreted by the IgA-positive cells could combine with the secretory piece produced by mucosal epithelial cells, then release

Y. Zhang et al. / Experimental Parasitology 141 (2014) 75–81

to the mucosal surface forming a protective layer. Furthermore, the results showed that chickens vaccinated with recombinant 3-1E protein plus AbISCOÒ-300 increased the positive expression rate of intestinal IgA in the present study (Table 2). In conclusion, all the findings in this report provide evidence that experimental immunization of chickens with E. acervulina recombinant 3-1E protein in combination with AbISCOÒ-300 adjuvant can provide protection against challenge infection of E. acervulina. The results also indicate that probably 5 lg is an appropriate dose for immunization. However, the precise molecular and cellular nature of the protective host responses induced by these complexes remains to be determined in the future. Acknowledgments This research was partially supported by the National Natural Science Foundation of China (31072128). References Agrawal, L., Haq, W., Hanson, C.V., Rao, D.N., 2003. Generating neutralizing antibodies, Th1 response and MHC non restricted immunogenicity of HIV-I env and gag peptides in liposomes and ISCOMs with in-built adjuvanticity. J. Immune Based Ther. Vaccines 1, 5. Arstila, T.P., Vainio, O., Lassila, O., 1994. Central role of CD4+ T cells in avian immune response. Poult. Sci. 73, 1019–1026. Berezin, V.E., Bogoyavlenskiy, A.P., Tolmacheva, V.P., Makhmudova, N.R., Khudyakova, S.S., Levandovskaya, S.V., Omirtaeva, E.S., Zaitceva, I.A., Tustikbaeva, G.B., Ermakova, O.S., Aleksyuk, P.G., Barfield, R.C., Danforth, H.D., Fetterer, R.H., 2008. Immunostimulating complexes incorporating Eimeria tenella antigens and plant saponins as effective delivery system for coccidia vaccine immunization. J. Parasitol. 94, 381–385. Berezin, V.E., Bogoyavlenskyi, A.P., Khudiakova, S.S., Alexuk, P.G., Omirtaeva, E.S., Zaitceva, I.A., Tustikbaeva, G.B., Barfield, R.C., Fetterer, R.H., 2010. Immunostimulatory complexes containing Eimeria tenella antigens and low toxicity plant saponins induce antibody response and provide protection from challenge in broiler chickens. Vet. Parasitol. 167, 28–35. Budec, M., Koko, V., Todorovic, V., Markovic, D., Postic, M., Drndarevic, N., Spasic, A., Mitrovic, O., 2007. Possible mechanism of acute effect of ethanol on intestinal IgA expression in rat. Int. Immunopharmacol. 7, 858–863. Ding, X., Lillehoj, H.S., Quiroz, M.A., Bevensee, E., Lillehoj, E.P., 2004. Protective immunity against Eimeria acervulina following in ovo immunization with a recombinant subunit vaccine and cytokine genes. Infect. Immun. 72, 6939– 6944. Gao, J., Zhang, H.J., Wu, S.G., Yu, S.H., Yoon, I., Moore, D., Gao, Y.P., Yan, H.J., Qi, G.H., 2009. Effect of Saccharomyces cerevisiae fermentation product on immune functions of broilers challenged with Eimeria tenella. Poult. Sci. 88, 2141–2151.

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