Avian Pathology

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Immune protection of microneme 7 (EmMIC7) against Eimeria maxima challenge in chickens Jingwei Huang, Zhenchao Zhang, Menghui Li, Xiaokai Song, Ruofeng Yan, Lixin Xu & Xiangrui Li To cite this article: Jingwei Huang, Zhenchao Zhang, Menghui Li, Xiaokai Song, Ruofeng Yan, Lixin Xu & Xiangrui Li (2015) Immune protection of microneme 7 (EmMIC7) against Eimeria maxima challenge in chickens, Avian Pathology, 44:5, 392-400, DOI: 10.1080/03079457.2015.1071780 To link to this article: http://dx.doi.org/10.1080/03079457.2015.1071780

Accepted online: 16 Jul 2015.Published online: 06 Oct 2015.

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Date: 13 October 2015, At: 19:28

Avian Pathology, 2015 Vol. 44, No. 5, 392–400, http://dx.doi.org/10.1080/03079457.2015.1071780

ORIGINAL ARTICLE

Immune protection of microneme 7 (EmMIC7) against Eimeria maxima challenge in chickens Jingwei Huang, Zhenchao Zhang, Menghui Li, Xiaokai Song, Ruofeng Yan, Lixin Xu, and Xiangrui Li*

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College of Veterinary Medicine, Nanjing Agriculture University, Nanjing, Jiangsu, People’s Republic of China

In the present study, the immune protective effects of recombinant microneme protein 7 of Eimeria maxima (rEmMIC7) and a DNA vaccine encoding this antigen (pVAX1-EmMIC7) on experimental challenge were evaluated. Two-week-old chickens were randomly divided into five groups. Experimental groups of chickens were immunized with 100 μg DNA vaccine pVAX1-MIC7 or 200 μg rEmMIC7, while control groups of chickens were injected with pVAX1 plasmid or sterile phosphate buffered saline (PBS). The results showed that the anti-EmMIC7 antibody titres in chickens of both rEmMIC7 and pVAX1-MIC7 groups were significantly higher as compared to PBS and pVAX1 control (P < .05). The splenocytes from both vaccinated groups of chickens displayed significantly greater proliferation response compared with the controls (P < .05). Serum from chickens immunized with pVAX1-MIC7 and rEmMIC7 displayed significantly high levels of interleukin-2, interferon-γ, IL-10, IL-17, tumour growth factor-β and IL-4 (P < .05) compared to those of negative controls. The challenge experiment results showed that both the recombinant antigen and the DNA vaccine could obviously alleviate jejunum lesions, body weight loss and enhance oocyst decrease ratio. The anti-coccidial index (ACI) of the pVAX1-MIC7 group was 167.84, higher than that of the recombinant MIC7 protein group, 167.10. Our data suggested that immunization with EmMIC7 was effective in imparting partial protection against E. maxima challenge in chickens and it could be an effective antigen candidate for the development of new vaccines against E. maxima.

Introduction Avian coccidiosis is caused by multiple species of the apicomplexan protozoa Eimeria, which invade discrete regions of the intestinal epithelium causing reduced feed conversion efficiency, leading to decreased body weight gain and even mortality (Vermeulen et al., 2001). Eimeria maxima, together with E. tenella and E. acervulina, is frequently considered to be one of the most economically relevant Eimeria spp. (Shirley et al., 2004). Prophylactic feeding of coccidiostat drugs is the major disease control method used in commercial settings. However, with increasing demands for high-protein meats and heightened consumer concerns over the use of antibiotics in poultry production, the search for alternative strategies against avian coccidiosis has intensified (Wallach et al., 1995). Live-attenuated vaccines protective against the seven Eimeria species that cause chicken coccidiosis are commercially available, but their applications are limited to a small part of the global poultry industry. In recent years, recombinant vaccines including subunit vaccines and DNA vaccines have been widely studied as a novel strategy to elicit protection against coccidiosis, and a number of promising antigens have been identified (Klotz et al., 2007; Subramanian et al., 2008; Zhu et al., 2012a).

*To whom correspondence should be addressed. E-mail: [email protected] (Received 21 January 2015; accepted 8 June 2015) © 2015 Houghton Trust Ltd

It has also been found that DNA vaccines can provoke both humoral and cell-mediated immune responses (Subramanian et al., 2008; Yang et al., 2010), and co-delivery of cytokines as adjuvants could enhance the potential for DNA vaccines or recombinant antigens to induce broad and long-lasting humoral and cellular immunity (Lillehoj et al., 2005; Song et al., 2010a). Micronemes (MICs) are secretory organelles that are located at the anterior end of the sporozoite and merozoite stages of all apicomplexan parasites (Witcombe et al., 2003). Microneme proteins are secreted in the early stages of the invasive process and participate in attachment to the host cell and subsequent formation of the connection with the parasite’s actinomyosin system (Morahan et al., 2009). Experiments have already proven that the major microneme adhesive repeat regions protein from E. tenella, microneme protein 3 (EtMIC3), was deployed at the parasite–host interface during the early stages of invasion (Lai et al., 2011). Thus, induction of neutralizing antibodies specific to one or several of these “invasion proteins” presents a rational approach in developing prophylactic vaccines. Eimeria maxima is one of the most prevalent Eimeria species in chickens. However, few genes of E. maxima have been reported and tested for their immunogenicity. In the current study, the gene of microneme protein 7 of

Microneme protein 7 of Eimeria maxima 393

E. maxima (EmMIC7) was cloned and the protective efficacies of both the recombinant protein and DNA vaccine encoding this antigen were evaluated.

Materials and Methods

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Parasites. Sporulated oocysts of E. maxima isolated from Jiangsu Province of China (JS) were propagated by repeated passage in three-week-old specific pathogen-free Chinese Yellow chickens at least every threemonths and were stored in 2.5% potassium dichromate solution at 4°C.

Chickens. Newly hatched day-old Chinese Yellow chickens were raised in a sterilized room under coccidia-free conditions until the end of the experiment. The room was fumigated with 40% formaldehyde solution and washed with 10% lime water. Chickens were screened periodically for their Eimeria infection status by microscopic examination of their faeces using the saturated salt flotation method every 12 h (Long & Rowell, 1975). The chickens were provided with coccidiostat-free feed and water ad libitum and shifted to the animal containment facility prior to challenge with virulent oocysts. The study was conducted following the guidelines of the Animal Ethics Committee, Nanjing Agriculture University, China. All experimental protocols were approved by the Science and Technology Agency of Jiangsu Province. The approval ID was SYXK (SU) 2010–0005.

Collection and purification of sporozoites. Sporozoites from sporulated E. maxima oocysts were purified on DE-52 anion exchange columns (GE Healthcare Life Sciences, Pittsburgh, PA, USA) using the protocol described previously (Klotz et al., 2007). Briefly, for each purification step 5 × 108 oocysts were sterilized for 10 min with 10% sodium hypochloride and washed with phosphate buffered saline (PBS) solution (pH 7.4) by centrifugation. Washed oocysts were disrupted by vigorous mixing with 10 ml Hanks balanced salt solution (HBSS) and 5 g glass beads (0.5 mm diameter). Sporocysts were recovered from glass beads with HBSS and subsequently sporozoites were released with excystation medium (0.15% trypsin, 2.5% sodium cholate) at 41°C. Sporozoites were washed and resuspended in 200 mM PBS pH 8.0 containing 1% glucose and purified on a DE-52 cellulose anion exchange column. The collected sporozoites were stored in liquid nitrogen before further use.

Soluble antigens of E. maxima. The preparation of soluble antigens of E. maxima was performed following the protocol described previously (Zhu et al., 2012b). Briefly, a count of 5 × 109 sporozoites was washed three times by centrifuge with 0.1 M PBS (pH 7.4) at 2000g for 10 min at 4°C. The pellet was dissolved in 2 ml PBS containing 0.5% Triton X-100 and was disrupted by ultrasound in an ice bath (200 W, work time 5 s, interval time 10 s, 50 cycles). After high-speed centrifugation (13,000g, 15 min) at 4°C, the supernatant proteins were separated and adjusted to 1 mg/ml with PBS and stored at −70°C until to be used for Western blot to analyse the native protein of the EmMIC7.

Chicken immune sera against E. maxima. For raising polyclonal sera against E. maxima, two-week-old birds were inoculated with 1.0 × 105 sporulated oocysts orally and one week later 1.0 × 105 sporulated oocysts were boosted by oral inoculation. These oocysts were deposited directly into the birds’ crop using a catheter. Blood was collected 10 days after the booster dose. Serum was separated from the blood by centrifugation and stored at −70°C until further use. Sera collected from chickens before Eimeria spp. infection was used as negative control.

Construction of prokaryotic expression vector of EmMIC7. Total RNA was prepared from sporulated oocysts of E. maxima using E.A.N.A.™ Total RNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA). The cDNA was synthesized by reverse transcription (RT) reaction using primers designed according to mRNA sequence of EmMIC7 gene (FR718975). Gene-specific primers were designed using the software “Primer Premier 5” (Premier Biosoft, Palo Alto, CA, USA). The primer set for MIC7 contained restriction enzyme sites EcoRI and XhoI in the forward (5′CCGGAATTCATGAGAAGCTTCGGGGCGAT-3′) and reverse primers

(5′-CCGCTCGAGTTACGCCGATTGCTCCACATT-3′) (sites for digestion by EcoRI and XhoI are underlined), respectively. Amplification was performed by an initial reaction at 95°C (3 min) followed by 30 cycles of 95° C (20 s), 58.5°C (20 s), 72°C (15 s), and final extension at 72°C (5 min) using the commercial kit 2× Phanta® EVO HS Master Mix (Vazyme biotech Co., Ltd., Nanjing, China). The amplification product was purified using “AxyPrep™ DNA Gel Extraction Kit” (Axygen, Union City, CA, USA) and then ligated to pUM 19-T cloning vector (Vazyme biotech Co., Ltd., Nanjing, China) according to the relevant manufacturer’s instructions. Selected clones containing the EmMIC7 sequence were checked by enzymatic digestion using the previously inserted restriction sites in the designed primers, followed by sequence confirmation by Invitrogen Biotech (Shanghai, PR China). The correct clones were then sub-cloned according to the designed restriction sites into pET-32a(+) vector (Merck Millipore, Billerica, MA, USA) to generate expression plasmid pET-32a(+)-MIC7. The recombinant plasmids were sequenced again to confirm that the inserts were in the correct reading frame.

Sequence analysis. Sequence similarity was analysed using BLASTP and BLASTX (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi). Microneme protein sequences were aligned using CLUSTALW1.8 8 (Thompson, 1994). The signal peptide, secondary structure and protein motifs were predicted using approaches accessible on the Internet: SignalP (http://www.cbs.dtu.dk/ services/SignalP/) (Petersen et al., 2011), TMHMM (http://www.cbs.dtu.dk/ services/TMHMM/) (Krogh et al., 2001), GPI Modification Site Prediction (http://mendel.imp.ac.at/sat/gpi/gpi_server.html) (Eisenhaber, 1999), O-glycosylation sites (http://www.cbs.dtu.dk/services/DictyOGlyc/) (Gupta et al., 1999), phosphorylation sites (http://www.cbs.dtu.dk/services/NetPhos/) (Blom et al., 2000), PSIpred (http://bioinf.cs.ucl.ac.uk/psipred/) (Buchan, 2013), Motifscan (http://www.myhits.isb-sib.ch/cgibin/motif_scan) (Pagni, 2007), respectively.

Expression of recombinant MIC7 protein. The plasmids pET-32a (+)-MIC7 generated as above were transformed into Escherichia coli BL21 (DE3). Recombinant protein expression was induced using isopropyl-β-d-thiogalactopyranoside (Sigma-Aldrich, St. Louis, MO, USA) at OD600 = 0.6. The induced bacterial cells were incubated for 5 h following which the cells were harvested by centrifugation. The cell pellet was lysed using lysozyme (10 μg/ml) (Sigma-Aldrich) followed by sonication and was then analysed by 12% (w/v) sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).

Purification of rMIC7. To purify the recombinant protein, induced E. coli cells were harvested by centrifugation and sonicated for 15 min on ice. After centrifugation at 10,000g, the supernatant was added to a Ni2+-nitrilotriacetic acid (Ni-NTA) column (GE Healthcare Life Sciences, Pittsburgh, Pennsylvania, USA) and purified according to the manufacturer’s instructions. An elution buffer (300 mM NaCl, 40 mM Na3PO4, pH 8.0) containing 500 mM of imidazole was utilized to wash the His-tagged proteins from the Ni-NTA column. The purity of the protein was confirmed by 12% SDSPAGE and the concentration of the purified protein was determined according to the Bradford procedure (Bradford, 1976), using bovine serum albumin as a standard. Endotoxin was removed from the protein samples employed in the in vivo trials using Thermo Scientific Pierce High Capacity Endotoxin Removal Spin Columns (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The purified protein was stored in aliquots at −70°C until further use.

Generation of antisera against recombinant MIC7 protein. To generate polyclonal antibody, about 0.3 mg of the purified recombinant MIC7 protein was mixed with Freund’s complete adjuvant at a 1:1 ratio and injected into Sprague-Dawley (SD) rats (Qualified Certification: SCXK 2008-004; Experimental Center of Jiangsu Province, PR China) subcutaneously in multiple places. The booster doses were the same to that of the primary immunization and delivered with Freund’s incomplete adjuvant at a 1:1 ratio. Three boosters were given weekly from the second week post the first immunization to the fourth. Finally, the serum was collected and stored at −70°C until used. Sera collected before protein injection was used as negative sera (Yan et al., 2013).

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Detection of the recombinant and native microneme proteins with Western blot. Samples including somatic extract (total soluble protein) of E. maxima sporozoites and the purified recombinant MIC7 protein were separated by SDS-PAGE. Then the proteins were transferred to nitrocellulose membrane (Merck Millipore) using “Semidry Transfer Cell” (Bio-rad, Hercules, CA, USA) according to the manufacturer’s instructions. After being blocked with 5% (w/v) skimmed milk powder in Tris Buffered Saline - Tween 20 (TBST), the membranes were incubated with primary antibodies (rat antisera and chicken antisera, respectively) for 2 h at 37°C (dilutions 1:200 to rat antisera, 1:100 to chicken antisera). Secondary antibodies, horseradish peroxidase (HRP)-conjugated goat anti-rat IgG and HRP-conjugated donkey-anti-chicken IgG (Sigma-Aldrich) were added, respectively. Finally, the bound antibody was detected using a 3,3′-diaminobenzidine tetrahydrochloride Kit (Boster Bio-technology, Wuhan, PR China) according to the manufacturer’s instructions.

Construction of eukaryotic expression vectors of EmMIC7. In order to construct pVAX1-MIC7 vector, the recombinant vector pUM 19-T-MIC7 generated previously was treated with EcoRI/XhoI. The digested fragment was directionally cloned into the pVAX1 vector using the associated ligation kit (Vazyme Biotech Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. Recombinant vector pVAX1-MIC7 was digested with the same restriction enzymes and sequenced by Invitrogen Biotech (Shanghai, PR China) for identification. The recombinant plasmid pVAX1-MIC7 employed as a DNA vaccine was prepared using a Qiagen EndoFree Plasmid Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. The eluted product was dissolved in Tris-EDTA (TE) buffer (pH 7.4) at a concentration of 1 mg/ml, and stored at −20°C until required.

Detection of the transcription and expression of proteins encoded by plasmid pVAX1-MIC7 in vivo. Two-week-old chickens were injected intramuscularly in the leg muscle with 100 μg of recombinant plasmid pVAX1MIC7. One week post-inoculation, injected tissues were collected for total RNA extraction. To remove contaminating genomic DNA or injected plasmid, all RNA samples were treated with RNase-free DNase I (TaKaRa, China). RT-PCR assays were performed with the cloning primer pairs from the EmMIC7 gene. The Polymerase Chain Reaction (PCR) products were detected by electrophoresis on 1% (w/v) agarose gel. Western blot analysis was performed as described previously (Xu et al., 2008). Briefly, seven days after vaccination, injected muscles were ground and treated with ice-cold Radio Immunoprecipitation Assay (RIPA) lysis buffer (Vazyme Biotech Co., Ltd., Nanjing, China). Meanwhile, muscles from the same site from non-injected and pVAX1 plasmid injected chickens were collected as controls. Proteins were separated by SDS-PAGE and then transferred to nitrocellulose membrane (Merck Millipore). The membrane was incubated with rat-anti-rMIC7 polyclonal antibody as primary antibody and HRP-conjugated goat anti-rat IgG (Sigma) as secondary antibody. The bound antibody was detected using a 3,3′-Diaminobenzidine Tetrahydrochloride Kit (Boster Bio-technology, Wuhan, PR China). Immunization experiment design. For the challenge experiment, twoweek-old chickens were weighed and randomly distributed into five groups of 35 chicks each as shown in Table 1. Experimental group chickens were respectively immunized with 200 μg recombinant MIC7 protein or 100 μg recombinant plasmid pVAX1-MIC7 by leg intramuscular injection. Challenged unvaccinated control group (positive control) and unchallenged unvaccinated control group (negative control) chickens were injected with only sterile PBS at the same injection site. Plasmid control group was given 100 μg of the pVAX1 plasmid alone. A booster immunization was given one week later with the same amount of components as the first immunization. Seven days post the second injection, all chickens of each group except the negative control group were inoculated orally with 1 × 105 sporulated oocysts of E. maxima JS. The oocysts were administered as a PBS suspension (1 ml), directly into the crop via a self-calibrated 1-ml micropipette. Negative control chickens were given PBS orally. Seven days after challenge, all chickens were weighed and euthanized for jejunum collection to determine the effects of immunization. For the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, 2-week-old chickens were divided into four groups of 10 chicks each. Plasmid and PBS control groups were also set as negative

controls. The immunization programme of the experimental groups was conducted as described above. Seven days after the booster immunization, chickens from each group were killed to separate the spleens for lymphocyte proliferation response determination. For the serum IgG determination, 2-week-old chickens were divided into four groups of 10 chicks each. Plasmid and PBS control groups were also set as negative controls. The immunization programme of the experimental groups was conducted as described above. The blood of chickens of each group were collected by cardiac puncture for determination of IgG antibody levels weekly, starting from the day of first immunization, ending at the fourth week post the booster immunization. The blood was allowed to clot for 1 h at 37°C and then overnight at 4°C. The serum was separated by centrifugation (800g, 10 min), and stored at −20°C until further use. For cytokine determination, two-week-old chickens were divided into four groups of 10 chicks each. Plasmid and PBS control groups were also set as negative controls. The immunization programme of the experimental groups was conducted as described above. Ten days post the booster immunization, the sera of chickens in each group were collected by cardiac puncture for cytokine determination. Determination of serum antibody levels. IgG antibody levels against EmMIC7 in the serum samples was determined by enzyme linked immunosorbent assay (ELISA) as described previously (Lillehoj et al., 2005). Briefly, flat-bottomed 96-well plates (Marxi-Sorp, Nunc, Denmark) were coated overnight at 4°C with 100 μl solution per well containing recombinant EmMIC7 (50 μg/ml) in 0.05 M carbonate buffer, pH 9.6. The plates were washed with 0.01 M PBS containing 0.05% Tween-20 (PBS-T) and blocked with 5% skim milk powder (SMP) in PBS-T for 2 h at 37°C. The plates were incubated for 2 h at 37°C with 100 μl of the serum samples diluted 1:50 in PBS-T with 5% SMP in duplicate. After three washes, the plates were incubated for 1 h at room temperature with 100 μl/well of HRP-conjugated donkey-anti-chicken IgG antibody (Sigma) diluted 1:1000 in 5% SMP in PBS-T. Colour development was carried out with 3,-3′,5,5′-tetramethylbenzidine (Sigma). The optical density at 450 nm (OD450) was determined with a microplate spectrophotometer. All serum samples were tested by ELISA at the same time under the same conditions, and the serum samples collected on each occasion were included on one plate.

Determination of cytokine concentrations. The concentration of interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-17 (IL-17) and tumour growth factor-β (TGF-β) in serum collected 10 days after the booster immunization of each group was detected by utilizing an indirect ELISA with the “Chicken Cytokine ELISA Quantization Kits” (catalog numbers: CSB-E08550Ch, CSBE06755Ch, CSB-E06756Ch, CSB-E12835C, CSB-E0467Ch, and CSBE09875Ch for IFN-γ, IL-2, IL-4, IL-10, IL-17 and TGF-β, respectively; CUSABIO, Wuhan, China) in duplicate, according to manufacturer’s instructions.

Detection of the proliferation of splenic lymphocytes in chickens immunized with EmMIC7. On day 7 post-second immunization, spleens were removed from 10 chickens and lymphocyte proliferation responses were determined by the MTT method described previously (Barta et al., 1992; Sasai et al., 2000). Briefly, single cell suspensions were prepared by gently pressing the freshly removed spleens with a stainless steel mesh (#60, 250 μm pore size) into a Petri dish containing calcium and magnesium free HBSS (Thermo Fisher Scientific, Waltham, MA, USA). After large clumps were allowed to settle, the cells were collected, washed with HBSS by centrifugation (500g, 10 min, 4°C), resuspended in HBSS, and centrifuged over lymphocyte separation solution (Haoyang Biotech, Tianjin, PR China) according to the manufacturer’s instructions. After three washes with PBS (pH 7.4), the concentration of the cells was adjusted to 5 × 106/ml. Each 100 μl were added to 96-well cell culture plate (Thermo Fisher Scientific). Con A (Sigma-Aldrich) was then added to a final concentration of 3 μg/ml and the plate was incubated for 24 h at 40.6°C in humidified air with 5% CO2. Twenty microlitre of 5 mg/ml MTT (Shengxing Bio, Nanjing, PR China) was added to each well and the plate was incubated for another 4 h in the same condition until purple precipitation was visible. Afterwards, detergent reagent (10% SDS, 0.04 N HCl) was added to each well and the plate was incubated for another 4 h in the same condition.

Microneme protein 7 of Eimeria maxima 395 Table 1. Effects of MIC7 against E. maxima challenge on immunorelevant parameters.

Groups Unchallenged control Challenged control pVAX1 control Recombinant EmMIC7 protein pVAX1-MIC7

Average body weight gain (mean ± SD)

Mean lesion scores (mean ± SD)

Oocyst output (×105) (mean ± SD)

Oocyst decrease ratio (%) A

ACI

A

77.95 ± 18.11 25.73 ± 15.83B 32.85 ± 17.01B 64.39 ± 21.31C

A

0.00 ± 0.00 2.63 ± 0.58B 2.24 ± 1.09B 1.45 ± 0.18C

A

0.00 ± 0.00 1.99 ± 1.35B 1.23 ± 0.79B 0.48 ± 0.71C

100 0B 38.19B 75.43C

200 66.71 99.74 167.10

64.11 ± 14.30C

1.31 ± 0.98C

0.44 ± 0.26C

75.38C

167.84

In each column, significant difference (P < .05) between numbers with different letters. No significant difference (P > .05) between numbers with the same letter.

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The optical density at 570 nm (OD570) was determined with microplate spectrophotometer.

Evaluation of immune protection of EmMIC7. The efficacy of immunization was evaluated on the basis of survival rate, lesion score, body weight gain, oocyst decrease ratio and ACI. Lesion score of the chickens from each group was investigated according to the method of Johnson & Reid, (1970). A microscope was used to examine scrapings for coccidia whenever there was doubt about the cause of a lesion. Oocyst counting was done using McMaster’s counting technique. Oocyst decrease ratio was calculated as follows: (the number of oocysts from positive control chickens − vaccinated chickens)/positive control chickens × 100%. ACI is a synthetic criterion for assessing the protective effect of a medicine or vaccine and is calculated as follows: (relative rate of weight gain + survival rate) − (lesion value + oocyst value).

Statistical analysis. All data were expressed as means ± SD and were performed using the SPSS Statistical Software (SPSS for windows 20.0, SPSS Inc., Chicago, IL, USA). Differences among the vaccinated and control groups were tested with the one-way Analysis of Variance (ANOVA) Duncan test (P < .05 was considered significantly different).

Results Cloning and sequence analysis of EmMIC7. The EmMIC7 amplification product was successfully isolated following PCR using the cDNA with gene-specific primers described above. The recovered PCR products were purified and successfully cloned into pUM 19-T cloning vector and confirmed by PCR and endonuclease digestion with EcoRI/ XhoI. Nucleic acid sequencing of the positive clones confirmed an insert of 519 bp. By analysis of the sequence, the open reading frame (ORF) was found to encode a protein of 172 amino acids with a predicted molecular mass of 18.24 kDa. The theoretical pI of the protein was 4.52. When compared with the sequences on the GenBank database, the results showed that the identity of the amino acid sequence of EmMIC7 to the microneme protein 7 of Eimeria maxima Houghton strain (CBX60037.1) was 100%. The amino acids 1–20 were predicted to be a signal peptide and the putative cleavage site between positions 20 and 21. However, no GPI anchor, transmembrane domain and O-glycosylation sites were found in the ORF of EmMIC7. Ten phosphorylation sites were predicted including eight serine sites and two threonine sites. No putative conserved domain was detected. Construction of both prokaryotic and eukaryotic expression vectors of MIC7. The ORF of MIC7 was successfully cloned into pET-32(+) and pVAX1 vectors using the method described above. Each target fragment was

detected by enzyme digestion and sequence analysis showed that the inserted fragment in the pET-32(+) and pVAX1 vectors was the 519 bp ORF of MIC7. Expression and purification of recombinant MIC7 protein. Recombinant plasmid conceiving cDNA fragment of MIC7 was expressed in vitro in E. coli BL21 (DE3). The recombinant protein was purified using the method described above. The peak amount of recombinant MIC7 protein was obtained 5 h after the beginning of isopropylβ-d-thiogalactopyranoside induction. The molecular weight of the expression fusion MIC7 protein was approximately 38 kDa on the SDS-PAGE gel. Since the fused protein of the pET-32a(+) vector was about 20 kDa, the molecular weight of the recombinant protein was about 18 kDa, identical to the calculated value (18.24 kDa). Detection of the recombinant and native MIC7 protein by Western blot. The results of the immunoblot assay (Figure 1) indicated that the recombinant MIC7 protein was recognized by immune sera of chickens infected with E. maxima, but the serum of uninfected chickens did not recognize recombinant MIC7 protein. Western blot analysis also showed that rat-anti-rMIC7 anti-serum bound to a band of about 16 kDa in the somatic E. maxima sporozoite extract (Figure 1). The unimmunized rat serum did not detect any protein within the somatic extract. The molecular weight of the detected natural MIC7 protein of E. maxima was slightly smaller than the calculated value. Identification of the expression of MIC7 by DNA vaccine in vivo. The results of RT-PCR indicated that the target fragment of MIC7 (approximately 519 bp) was detected from muscle RNA samples of chickens injected with pVAX1MIC7. No specific bands were detected in non-injected control and pVAX1 plasmid control samples. Western blotting of the muscle of chickens injected with pVAX1-MIC7 revealed a prominent band of 16 kDa, which indicated the expression of MIC7 gene. No corresponding band was detected in the muscle of chickens injected with pVAX1. IgG and cytokine levels in sera of immunized chickens. The serum EmMIC7-specific IgG levels in chickens following vaccination with both rEmMIC7 protein and pVAX1MIC7 are shown in Figure 2. The anti-EmMIC7 antibody titres of both rEmMIC7 and pVAX1-MIC7 groups were significantly higher as compared to PBS and pVAX1 control (P < .05). Antibody titres increased considerably in week 1 and started to decrease by week 2 post-second immunization.

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Figure 1. Immunoblot for recombinant and native EmMIC7 protein. Lane M, standard protein molecular weight marker; lane 1, recombinant MIC7 protein (pET-32a(+) 20 kDa fusion proteins of Trx tag, His tag, thrombin, S tag and enterokinase included) probed by serum of unimmunized chickens as primary antibody; lane 2, recombinant MIC7 protein (pET-32a(+) 20 kDa fusion proteins of Trx tag, His tag, thrombin, S tag and enterokinase included) probed by serum from chickens experimentally infected with E. maxima as primary antibody; lane 3, somatic extract of E. maxima sporozoites probed by serum of unimmunized rats as primary antibody. Lane 4, somatic extract of E. maxima sporozoites probed by rat-anti-rEmMIC7 antisera as primary antibody.

Non-specific antibody was detected in PBS control and pVAX1 control groups throughout the experiment (Figure 2). As depicted in Figure 3, serum from chickens immunized with pVAX1-MIC7 and rEmMIC7 protein showed significantly high levels of IL-2 and IFN-γ (P < .05) compared to those of controls. Within the same time, significantly high levels of IL-10 and IL-17 were also observed in immunized chickens. In the cases of TGF-β and IL-4, the enhanced levels in vaccinated chickens were also significant (P < .05), but modest. Proliferation responses of splenocytes. As shown in Figure 4, splenocytes from both vaccinated groups of chickens displayed significantly greater proliferation compared with pVAX1 and PBS control (P < .05). However no significant differences were observed between the vaccinated groups. Protective effects of MIC7 against E. maxima. The immunization efficacies of the vaccines are shown in Table 1. No chicken died from E. maxima coccidial challenge in any group. Chickens immunized with DNA vaccine pVAX1MIC7 and recombinant MIC7 protein displayed significantly enhanced weight gains, lower lesion scores, lower

oocyst production and higher oocyst decrease ratio compared with the chickens in pVAX1 and challenged control group (P < .05). The ACIs of both the rEmMIC7 and pVAX1-MIC7 vaccinated groups were more than 160, which indicated medium protective immunity against E. maxima infection (Chapman & Shirley, 1989).

Discussion In the current study, the immune protective effects of recombinant EmMIC7 protein and DNA vaccines were evaluated against E. maxima challenge. Our data suggest that immunization with EmMIC7 could increase serum IgG antibody titres and enhance the expression of cytokines including IL-2, IFN-γ, IL-10, IL-17, TGF-β and IL-4. The splenocytes from both vaccinated groups of chickens displayed significantly greater proliferation compared with pVAX1 and PBS control. The results of the animal experiments demonstrated that immunization with EmMIC7 could provide ACIs of more than 167. These results showed that immunization with EmMIC7 could induce partial protection against live E. maxima infection.

Figure 2. EmMIC7-specific IgG levels in chickens’ serum. Each group of chickens was immunized with 100 μg of pVAX1-MIC7, 200 μg of rEmMIC7 protein, 100 μg of pVAX1 or sterile PBS solution, respectively. One week later, a booster immunization was given with the same amount of components as the primary immunization. The blood from each group of chickens were collected for determination of IgG levels weekly using ELISA with respect to absorbance at 450 nm, starting from the day of first immunization, ending at the fourth week post the booster immunization. The concentrations of the IgG levels are expressed as mean ± SD.

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Figure 3. Serum cytokines of chickens vaccinated or unvaccinated using EmMIC7. Each group of chickens was immunized with 100 μg of pVAX1-MIC7, 200 μg of rEmMIC7 protein, 100 μg of pVAX1 or sterile PBS solution, respectively. One week later, a booster immunization was given with the same amount of components as the primary immunization. On day 10 post-second immunization, blood from each group of chickens was collected and the serum was separated for cytokine determination using ELISA with respect to the absorbance at 450 nm. The concentrations of IFN-γ, IL-2, IL-4, IL-10 and TGF-β are expressed as mean ± SD in pg/ml, IL-17 concentration (mean ± SD) in ng/ ml. Bars with different letters were considered to be significantly different (P < .05). The concentration of (a) IFN-γ; (b) TGF-β; (c) IL-2; (d) IL-4; (e) IL-10 and ( f ) IL-17.

The role of humoral immunity during immune responses protective against coccidiosis is debatable and is usually considered to play a minor role (Yun et al., 2000). However, recent studies have demonstrated that antibodies do play an important role in immunity against coccidiosis (Belli et al., 2004; Constantinoiu et al., 2008; Hoan et al., 2014). It was reported that the MIC proteins of Eimeria participated in the invasion of the parasite to the

host cell (Lai et al., 2011). Thus, antibody against a MIC protein might block invasion of the specific coccidian into the host and play an important role in the protection against Eimeria infection. In the present study, we demonstrated that the antibody titres of chickens vaccinated with pVAX1-MIC7 and rEmMIC7 were significantly higher than those of PBS and pVAX1 control during weeks 1–5 post the primary immunization (P < .05). It intensified the

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Figure 4. Proliferation responses of splenic lymphocytes from chickens immunized with EmMIC7. Each group of chickens was immunized with 100 μg of pVAX1-MIC7, 200 μg of rEmMIC7 protein, 100 μg of pVAX1 or sterile PBS solution, respectively. One week later, a booster immunization was given with the same amount of components as the primary immunization. On day 7 post-second immunization, spleens were removed from 10 chickens and lymphocyte proliferation was determined by the MTT method with respect to the absorbance at 570 nm. Bars with different letters were significantly different (P < .05).

function of antibody in the immune protection against Eimeria infection. The Th1-type cytokines, such as IFN-γ and IL-2, are responsible for classic cell-mediated functions and seem to be dominant during coccidiosis (Lowenthal et al., 1997). It was reported that IFN-γ and IL-2 transcript levels of chickens vaccinated with DNA vaccines were significantly increased and the chickens immunized with DNA vaccines pVAX1-LDH-IL-2 and pVAX1-LDH-IFN-γ obtained higher oocyst decrease ratio and ACIs than chickens immunized with pVAX1-LDH (lactate dehydrogenase, LDH) (Song et al., 2010b). In the current study, we also observed the concentrations of IFN-γ of the vaccinated groups were 12–16 fold greater than that of the control groups, meanwhile, the concentrations of IL-2 of the vaccinated groups were 3.5–4.5 fold greater than that of the control groups. The previous study and our data together confirm that IFN-γ and IL-2 might play a role in the immunization against coccidiosis. It is believed that the cell-mediated immune response plays a major role in conferring protective immunity against coccidiosis (Subramanian et al., 2008). Splenocytes from E. tenella immune chickens can inhibit intra-cellular development of E. tenella in chicken kidney cells in vitro (Miller et al., 1994). In the present study, our data showed that splenocytes from chickens vaccinated with pVAX1MIC7 or rEmMIC7 protein displayed significantly greater proliferation compared with the negative controls (P < .05). These results indicated that the EmMIC7 gene could induce cell-based immune responses against the Eimeria antigen. IL-4 is a typical Th2 cytokine and known to regulate humoral immunity more effectively as a helper for B-cell activation (Inagaki-Ohara et al., 2006). It has been demonstrated that IL-4 plays an important role in the response to intestinal parasite infection (Fallon et al., 2002). The results in our study showed that IL-4 concentrations of the vaccinated groups were significantly higher compared with the control groups. These results, together with the high antibody levels in the present study, indicated that IL-4 was associated with the protective immunity to coccidiosis. IL-17 is produced by Th17 cells and plays critical roles in immunological control of a variety of infectious diseases

(Zhang et al., 2013). Th17-related cytokines were induced during oral experimental E. tenella infection, and faecal oocyst shedding and caecal lesion scores could be reduced by using IL-17 antibody neutralization (Zhang et al., 2013). Further, IL-17 contributed to immunopathology during E. tenella infection and the elevated IL-17 might be harmful to the host. However, significantly higher levels of IL-17 in chickens vaccinated with DNA vaccines or subunit vaccines has also been observed (Song et al., 2010a; Hoan et al., 2014). In the current study, IL-17 concentrations in the vaccinated groups were at least 3.5-fold greater than those of the unvaccinated groups (P < .05). Thus, the real functions of IL-17 in the immunity to coccidiosis should be further researched. The TGF-β family of cytokines is biofunctional molecules exhibiting both growth promoting and growth inhibitory properties and can inhibit the proliferation of T- and B-lymphocytes (Kehrl et al., 1986; Choi et al., 1999). TGF-β mRNA transcription was increased in both spleen and intestine following experimental E. acervulina infection (Choi et al., 1999). In previous vaccination trials against coccidiosis, a significantly higher level of TGF-β was observed in chickens immunized with recombinant EbAMA1 protein and recombinant plasmid pVAX1-EbAMA1 (Hoan et al., 2014). The same results were also obtained using other vaccines against Eimeria (Song et al., 2010b; Zhu et al., 2012a). In the present report, we observed significantly higher levels of TGF-β in chickens immunized with recombinant EmMIC7 protein and recombinant plasmid pVAX1-MIC7. The exact functions of TGF-β in the infection and immune responses to Eimeria, especially the inhibitory effects of TGF-β on T cells, B cells and the protection of the vaccines, need to be further investigated. The predominant effect of IL-10 in mammals is to modify immune responses through direct effects on many cell types, including T cells, B cells, antigen presenting cells (APCs), and Natural Killer cells (NK), promoting the development of Th2 responses (Mocellin et al., 2003). In the present study, we observed significantly higher concentrations of IL-10 in groups vaccinated with pVAX1-MIC7 and rEmMIC7 compared with negative control groups. However, previous studies suggested that IL-10 might play a crucial role to block the development of strong IFN-γ-

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driven responses during infection of E. maxima (Rothwell et al., 2004). Thus, the exact functions of IL-10 in the protection against coccidiosis should be further studied. In the present study, an MTT assay was employed to investigate the effects of EmMIC7 proteins on the functions of lymphocytes in vivo. Con A is a plant mitogen and is known for its ability to stimulate T cell subsets giving rise to functional distinct T cell populations. The response abilities of the cells to the stimulation of Con A usually reflect the functional potential of the cells (Dwyer & Johnson, 1981). In this study, the splenocytes from both vaccinated groups of chickens displayed significantly greater proliferation compared with pVAX1 and PBS control (P < .05). It indicated that the EmMIC7 proteins directly immunized or expressed by the DNA vaccine, could enhance the proliferation of T cells to Con A stimulation and thus increase the functional potential of the cells. The successful construction, transcription and expression of the DNA vaccine pVAX1-MIC7 was confirmed using RTPCR and Western blotting. In previous research, two methods have been applied to confirm the successful application of DNA vaccines based on the pVAX1 vector. One has been injection of the recombinant vector into animal muscle (Song et al., 2010b; Shah et al., 2011). Another has been transfection of the vector into cultured cells (Liu et al., 2013; Hassan et al., 2014). In our present study, we used the former to confirm successful construction of the DNA vaccine. The results of RT-PCR and Western blotting confirmed that the DNA vaccine pVAX1-MIC7 could be transcribed and translated in chicken muscle. In the current work, sera raised against rEmMIC7 protein recognized a band of about 16 kDa in the somatic extract of E. maxima sporozoites. In the detection of the expression of DNA vaccine pVAX1-MIC7, we also observed that a band of 16 kDa could be recognized by the rat-anti-rEmMIC7 serum. These results suggest that the native MIC7 was approximately 16 kDa, slightly smaller than the calculated size of 18.24 kDa. Sequence analysis of EmMIC7 suggested that a signal peptide of 20 amino acids and 10 phosphorylation sites were present in this ORF. Usually, phosphorylation should enlarge the molecular weight of the peptide. Therefore, one of the possibilities resulted in the smaller native EmMIC7 is the removal of the signal peptide. However, it should be further investigated. Immunization with the pET-32a(+) tag-protein alone might have influenced host immunity. However, in previous studies, it has been well documented that immunization of the pET-32a(+) tag-protein alone could not induce protective immunity against Eimeria infection (Jang et al., 2010; Zhu et al., 2012b; Zhang et al., 2014). Therefore, this group was not included in order to reduce experimental cost and chicken usage. In conclusion, our data demonstrates that immunization with EmMIC7 can induce both humoral and cell-mediated immune responses against E. maxima and result in partial protection against E. maxima challenge in chickens. Thus, EmMIC7 might be an effective antigen candidate for the development of a new vaccine against E. maxima.

Acknowledgements We thank Mr Ibrahim A. Hassan from the College of Veterinary Medicine, Nanjing Agriculture University for reading this manuscript.

Funding This work was funded by a grant from the National Natural Science Foundation of PR China [grant no. 31372428] and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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