Eimeria maxima microneme protein 2 delivered as DNA vaccine and recombinant protein induces immunity against experimental homogenous challenge

Parasitology International 64 (2015) 408–416

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Parasitology International journal homepage: www.elsevier.com/locate/parint

Eimeria maxima microneme protein 2 delivered as DNA vaccine and recombinant protein induces immunity against experimental homogenous challenge Jingwei Huang, Zhenchao Zhang, Menghui Li, Xiaokai Song, Ruofeng Yan, Lixin Xu, Xiangrui Li ⁎ College of Veterinary Medicine, Nanjing Agriculture University, Nanjing, Jiangsu 210095, PR China

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Article history: Received 10 February 2015 Received in revised form 29 May 2015 Accepted 9 June 2015 Available online 11 June 2015 Keywords: Eimeria maxima Microneme protein 2 Cytokines Lymphocyte proliferation

a b s t r a c t E. maxima is one of the seven species of Eimeria that infects chicken. Until now, only a few antigenic genes of E. maxima have been reported. In the present study, the immune protective effects against E. maxima challenge of recombinant protein and DNA vaccine encoding EmMIC2 were evaluated. Two-week-old chickens were randomly divided into five groups. The experimental group of chickens was immunized with 100 μg DNA vaccine pVAX1-MIC2 or 200 μg rEmMIC2 protein while the control group of chickens was injected with pVAX1 plasmid or sterile PBS. The results showed that the anti-EmMIC2 antibody titers of both rEmMIC2 protein and pVAX1MIC2 groups were significantly higher as compared to PBS and pVAX1 control (P b 0.05). The splenocytes from both vaccinated groups of chickens displayed significantly greater proliferation compared with the controls (P b 0.05). Serum from chickens immunized with pVAX1-MIC2 and rEmMIC2 protein displayed significantly high levels of IL-2, IFN-γ, IL-10, IL-17, TGF-β and IL-4 (P b 0.05) compared to those of negative controls. The challenge experiment results showed that both the recombinant protein and the DNA vaccine could obviously alleviate jejunum lesions, body weight loss, increase oocyst, decrease ratio and provide ACIs of more than 165. All the above results suggested that immunization with EmMIC2 was effective in imparting partial protection against E. maxima challenge and it could be an effective antigen candidate for the development of new vaccines against E. maxima. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Coccidiosis, caused by parasites of the genus Eimeria, remains a major health problem in poultry farming and has a high economical impact on animal husbandry [1]. Eimeria maxima, together with Eimeria tenella and Eimeria acervulina, is frequently considered to be one of the most economically relevant Eimeria spp. [2]. Presently, chemotherapeutic treatment remains the most cost effective means of controlling the disease, however, the development of resistance to anticoccidial drugs and increasing public pressure to limit the use of chemicals in animal feed continues to drive the development of anti-coccidial vaccines [3]. One such alternative for the control of coccidiosis is live vaccines. However, live vaccines offer some disadvantages such as relatively high production costs, high reproductive potential of virulent Eimeria vaccine strains and possible reconstitution of attenuated Eimeria vaccine strains [4]. In addition, due to antigenic variation of strain populations, vaccine efficacy can differ geographically [5]. These drawbacks have driven the developments of new control strategies. ⁎ Corresponding author at: College of Veterinary Medicine, Nanjing Agriculture University, 1 Weigang, Nanjing, Jiangsu 210095, PR China. E-mail address: [email protected] (X. Li).

http://dx.doi.org/10.1016/j.parint.2015.06.002 1383-5769/© 2015 Elsevier Ireland Ltd. All rights reserved.

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 and used for experimental studies [6–8]. It has also been found that DNA vaccines or recombinant antigen can provoke both humoral and cell-mediated immune responses [9,10], and co-delivery of cytokines as adjuvants could enhance the potential for DNA vaccines or recombinant antigen to induce broad and long-lasting humoral and cellular immunity [11]. Micronemes are secretory organelles that are located at the anterior end of invasive stages of all apicomplexan parasites [12]. Infection by apicomplexans is established in the host by rapid and forced invasion of host cells using a multiple process [13]. Microneme proteins were secreted in the early stages of this process and participated in attachment to the host cell and subsequent formation of the connection with the parasite actinomyosin system, thereby providing the platform from which to drive invasion [14]. Experiments had already been conducted to prove that the major MARR (microneme adhesive repeat regions) protein from E. tenella, EtMIC3, is deployed at the parasite–host interface during the early stages of invasion [15]. Thus, induction of the neutralizing antibodies to one or several of these ‘invasion proteins’ presents a rational approach in developing a prophylactic vaccine.

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Eimeria maxima. is one of the most prevalent Eimeria species in chicken. However, few genes of E. maxima have been reported and tested for their immunogenicity. In the current study, a microneme gene of E. maxima, EmMIC2, was cloned and the protective efficacies of both the recombinant protein and DNA vaccine encoding this antigen were evaluated. 2. Materials and methods 2.1. Parasites and chickens Sporulated oocysts of E. maxima isolated from Jiangsu Province of China (JS) were propagated by repeated passages in 3-week-old specific pathogen-free Chinese Yellow chickens at least every 3-month intervals and were stored in 2.5% potassium dichromate solution at 4 °C. Newly hatched day-old Chinese Yellow chickens were raised in a sterilized room under coccidia-free conditions until the end of the experiment. Chickens were screened periodically for their Eimeria infection status by microscopic examination of their feces. 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 is SYXK (SU) 2010-0005. 2.2. Collection and purification of sporozoites Sporozoites from E. maxima oocysts were purified on DE-52 anion exchange columns using the protocol described previously [4]. Briefly, for each purification step, 5 × 108 oocysts were sterilized for 10 min with 10% sodium hypochloride and floated by centrifugation. Washed oocysts were disrupted by vigorous mixing with 10 ml Hanks balanced salt solution (HBSS) and 2 g glass beads (0.2 mm diameter, Omega BioTek, USA). 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 phosphate buffered saline pH 8.0 (PBS) containing 1% glucose and purified on a DE-52 cellulose anion exchange column (Whatman). The collected sporozoites were stored in liquid nitrogen before further use. 2.3. Soluble antigens of E. maxima The preparation of soluble antigens of E. maxima was performed following the protocol. A count of 5 × 109 sporozoites was washed three times by centrifuge with 0.1 M PBS (pH 7.4) at 2000 g for 10 min at 4 °C. The pellet was dissolved in 2 ml PBS containing 0.5% Triton X100 and was disrupted by ultrasound in ice bath (200 W, work time 5 s, interval time 10 s, 50 cycles). After high-speed centrifugation, 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 analysis the native protein of the EmMIC2. 2.4. 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 1 week later 1.0 × 105 sporulated oocysts were boosted with 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 from chickens without Eimeria spp. infection was used as negative control.

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2.5. Construction of prokaryotic expression vector of EmMIC2 Total RNA was prepared from sporulated oocysts of E. maxima using E.A.N.A. Total RNA Kit (Omega Biotek Inc., USA). The cDNA was synthesized by reverse transcription (RT) reaction using primers designed according to mRNA sequence of EmMIC2 gene (FR718971.1). Genespecific primers were designed using the software ‘Primer Premier 5’. The primer set for EmMIC2 contained restriction enzyme sites EcoRI and XhoI in forward primer (5′-CCGGAATTCATGGCTCGCGCCCTTTCAT-3′) and reverse primer (5′-CCCCTCGAGCTAGGAGCTGACCGATGTTGTGG-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 (30 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 a ‘AxyPrep™ DNA Gel Extraction kit’ (Axygen, USA) according to the manufacturer's instructions. And then they were ligated to pUM 19-T cloning vector (Vazyme biotech Co., Ltd., Nanjing, China) according to the manufacturer's instructions. Selected clones of EmMIC2 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 pET32a(+) vector (Novagen, USA) to generate expression plasmid pET32a(+)-MIC2. The recombinant plasmids were sequenced again to confirm that the inserts were in the correct reading frame. Sequence similarity was analyzed using the BLASTP and BLASTX (http://blast. ncbi.nlm.nih.gov/Blast.cgi). 2.6. Expression and purification of recombinant EmMIC2 protein The plasmids pET-32a(+)-MIC2 previously generated were transformed into Escherichia coli BL21 (DE3). Recombinant protein expression was induced using isopropyl-β-D-thiogalactopyranoside (IPTG; Sigma-Aldrich, USA) at OD 600 = 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) (SigmaAldrich, USA) followed by sonication and was then analyzed by 12% (w/v) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). To purify the recombinant protein, induced E. coli cells were harvested by centrifugation and sonicated for 15 min on ice. After centrifugation at 10,000 g, the supernatant was added to a Ni2+-nitrilotriacetic acid (NiNTA) column (GE Healthcare, USA) and purified according to the manufacturer's instructions. An elution buffer (500 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 detected by 12% SDS-PAGE and the concentration of the purified protein was determined according to the Bradford procedure [16], using bovine serum albumin (BSA) as a standard. The protein samples used for animal experiment were rid of endotoxin by Thermo Scientific Pierce High Capacity Endotoxin Removal Spin Columns according to the manufacturer's instructions (https://www.piercenet.com/instructions/2162373.pdf). The purified protein was stored in aliquots at −70 °C until further use. 2.7. Generation of anti-sera against recombinant MIC2 protein To generate polyclonal antibody, about 0.3 mg of the purified recombinant MIC2 protein was mixed with Freund's complete adjuvant of at 1:1 ratio and injected into SD rats (Qualified Certification: SCXK 2008004; Experimental Center of Jiangsu Province, PR China) subcutaneously in multiple places on the back. The booster doses were all delivered with Freund's incomplete adjuvant at 1:1 ratio. The first booster was performed 2 weeks post the first immunization, the 2nd 3 weeks and the 3rd 4 weeks. Finally, the rat anti-serum was collected and stored at −70 °C until used. Sera collected before protein injection was used as negative sera [17].

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2.8. Detection of the recombinant and native microneme proteins with western-blot Samples including somatic extract of E. maxima sporozoites and the purified recombinant EmMIC2 protein were separated by SDS-PAGE. Then the proteins were transferred to nitrocellulose membrane (Millipore, USA). After being blocked with 5% (w/v) skimmed milk powder in TBS-Tween 20 (TBST), the membranes were incubated with first antibodies (rat antisera and chicken antisera, respectively) for 2 h at 37 °C (dilutions 1:200 to rat antisera, 1:100 to chicken antisera). Second antibodies, horseradish peroxidase (HRP)-conjugated goat anti rat IgG, and HRP-conjugated donkey anti-chicken IgG (Sigma, USA) were added, respectively. Finally, the bound antibody was detected using 3, 3′-diaminobenzidine tetrahydrochloride (DAB) kit (Tiangen Biotech, Beijing, PR China) according to the manufacturer's instructions.

2.9. Construction of eukaryotic expression vectors of EmMIC2 In order to construct pVAX1-MIC2 vector, the recombinant vector pUM 19-T-MIC2 previously generated was treated with EcoRI/XhoI. The digested fragment was directionally cloned into the pVAX1 vector (Invitrogen, Life Technologies) using the ligation kit (Vazyme Biotech Co., Ltd., Nanjing, China) according to the manufacturer's instructions. Recombinant vector pVAX1-MIC2 was digested with the same restriction enzymes and sequenced by Invitrogen Biotech (Shanghai, PR China) for identification. The recombinant plasmid pVAX1-MIC2 acting as DNA vaccine was prepared using Qiagen EndoFree Plasmid Kit (Qiagen, USA), according to the manufacturer's instructions. The eluted product was dissolved in PBS (pH 7.4) at a concentration of 1 mg/ml, and stored at −20 °C until required.

2.11. Immunization assays Two-week-old chickens were weighed and randomly distributed into five groups of 65 chicks each as shown in Table 1. Experimental group chickens were respectively immunized with 200 μg rEmMIC2 protein and 100 μg recombinant plasmids pVAX1-MIC2 by a leg intramuscular injection. The challenged control group (positive control) and unchallenged control group (negative control) chickens were injected with only sterile PBS at the same injection site. Plasmid control group was given 100 μg of pVAX1 plasmid alone each chicken. A booster immunization was given one week later with the same amount of components as the first immunization. Seven days post the second injection, 35 chickens of each group except the unchallenged control group were inoculated orally with 1 × 105 sporulated oocysts of E. maxima JS. Unchallenged control chickens were given PBS orally. Seven days after challenge, all chickens were weighed and euthanized for the jejunum collection to determine the effects of immunization. The rest 30 unchallenged chickens in each group were raised in another coccidian-free room. Ten chickens of each group were killed 7 days after the booster immunization by cardiac puncture to separate the spleens for lymphocyte proliferation response determination. The blood of other 10 unchallenged chickens of each group was collected by cardiac puncture for determination of IgG antibody levels weekly, starting from the day of first immunization, ending at the 4th 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 (800 g, 10 min), and stored at − 20 °C until further use. The sera of the rest 10 unchallenged chickens in each group were collected by cardiac puncture for determination of cytokines 10 days post the booster immunization. 2.12. Determination of serum antibody level and cytokine concentration

2.10. Detection of the expression of proteins encoded by plasmid pVAX1MIC2 in vivo by RT-PCR assay and western blot analysis Two-week-old chickens were injected intramuscularly (IM) in leg muscle with 100 μg of recombinant plasmid pVAX1-MIC2. One week post-inoculation, injected tissues were collected for total RNA extraction. To remove contaminating genomic DNA or plasmids injected, all RNA samples were treated with RNase-free DNase I (Vazyme Biotech Co., Ltd., Nanjing, China). RT-PCR assays were performed with cloning primer pairs of EmMIC2 gene. The PCR products were detected by electrophoresis on 1% agarose gel. Western blot analysis was performed as described previously [18]. Briefly, seven days after vaccination, injected muscles were grinded and treated with ice-cold RIPA solution (Vazyme Biotech Co., Ltd., Nanjing, China). Meanwhile, the same site muscles from non-injected and pVAX1 plasmid injected chickens were collected as controls. Proteins were separated by SDS-PAGE and then transferred to nitrocellulose membrane (Millipore, USA). The membrane was incubated with rat anti-rEmMIC2 polyclonal antibody as primary antibody and horseradish peroxidase (HRP)-conjugated goat anti rat IgG (Sigma) as second antibody. The bound antibody was detected using DAB kit (Boster BioTechnology, Wuhan, PR China).

The IgG antibody levels against E. maxima soluble antigen in the serum samples were determined by ELISA as described previously [7]. Briefly, flat-bottomed 96-well plates (Marxi-Sorp, Nunc, Denmark) were coated overnight at 4 °C with 100 μl solution per well containing rEmMIC2 (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 1% 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, USA) diluted 1:1000 in 5% SMP in PBS-T. Color development was carried out with 3, -3′, 5, 5′-tetramethylbenzidine (TMB) (Sigma). The optical density at 450 nm (OD450) was determined with 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. The concentration of interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-17 (IL-17), and tumor growth factor-β (TGF-β) in serum collected ten days after the booster immunization of each group was detected by utilizing an

Table 1 Effects of MIC-2 against E. maxima challenge on different parameters. Groups

Average body weight gain (mean ± SD)

Mean lesion scores (mean ± SD)

Oocyst output (×105) (mean ± SD)

Oocyst decrease ratio (%)

Anti-coccidial index

Unchallenged control Challenged control pVAX1 control Recombinant EmMIC2 protein pVAX1-MIC2

79.80 ± 12.89a 29.91 ± 31.71b 35.23 ± 23.63b 63.93 ± 38.59c 67.16 ± 20.63c

0.00 ± 0.00a 2.96 ± 0.35b 2.81 ± 0.42b 1.33 ± 0.25c 1.30 ± 0.85c

0.00 ± 0.00a 2.89 ± 0.16b 2.62 ± 0.34b 0.58 ± 0.21c 0.49 ± 0.25c

100a 0b 9.34b 79.93c 77.89c

200 67.88 76.04 165.81 170.16

Note: In each column, significant difference (P b 0.05) between numbers with different letters. No significant difference (P N 0.05) between numbers with the same letter.

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indirect ELISA with the ‘Chicken Cytokine ELISA Quantization Kits’ (catalog numbers: CSB-E08550Ch, CSB-E06755Ch , CSB-E06756Ch, CSBE12835C, CSB-E0467Ch, and CSB-E09875Ch for IFN-γ, IL-2, IL-4, IL-10, IL-17 and TGF-β respectively; CUSABIO, China) in duplicate, according to the manufacturer's instructions. 2.13. Detection of the proliferation of splenic lymphocytes in chickens immunized with EmMIC2 On day 7 post-second immunization, the spleens were removed from 10 chickens and lymphocyte proliferation responses were determined by MTT method described previously [19,20]. 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 Hank's balanced salt solution (HBSS, GIBCO BRL, USA). After large clumps were allowed to settle, the cells were collected, washed with HBSS by centrifugation (500 g, 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 phosphate buffered saline (PBS, pH 7.4), the concentration of the cells was adjusted to 5 × 106/ml. Each 100 μl was added to 96-well cell culture plate (Thermo Fisher Scientific Inc., USA). ConA (Sigma, USA) was then added to final concentration of 3 μg/ml and the plate was incubated for 24 h at 40.6 °C in humidified air with 5% CO2. 20 μl 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. Afterward, 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. The optical density at 570 nm (OD570) was determined with microplate spectrophotometer.

Fig. 1. Agarose gel electrophoresis of PCR product of EmMIC2 gene, endonuclease digestion of recombinant plasmid pET-32a(+)-MIC2 and p VAX1–MIC2. Lane M, DNA marker DL5000; Lane 1, PCR product of EmMIC2. Lane 2, pET-32a(+)-MIC2 digested by EcoRI and XhoI. Lane 3, pVAX1-MIC2 digested by EcoRI and XhoI.

2.14. Evaluation of immune protection of EmMIC2 The efficacy of immunization was evaluated on the basis of survival rate, lesion score, body weight gain, oocyst decrease ratio and anticoccidial index (ACI). Survival rate was estimated by the number of surviving chickens divided by the number of initial chickens at the time of challenge. Lesion score of the chickens from each group was investigated according to the method [21]. 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). 2.15. 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 ANOVA Duncan test (P b 0.05 was considered significantly different). 3. Results 3.1. Cloning of EmMIC2 The amplification product of EmMIC2 was successfully isolated following PCR using the cDNA with gene-specific primers described previously (Fig. 1). The recovered PCR products were purified and successfully cloned into pUM 19-T cloning vector which was confirmed by PCR and endonuclease digestion with EcoRI/XhoI. Nucleic acid sequencing of the positive clones of MIC2 contained an insert of 888 bp.

Fig. 2. Purified recombinant MIC2 protein resolved on a SDS-PAGE, stained with Coomassie brilliant blue. The purified recombinant MIC2 protein is seen as a band of approximately 50.5 kDa.

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The Blastp results showed that the identity of the EmMIC2 amino acid sequence to the microneme protein 2 of E. maxima Houghton strain (CBX60033.1) was 100%.

3.2. Construction of both prokaryotic and eukaryotic expression vectors of MIC2 The ORF of MIC2 was successfully cloned into pET-32a(+) and pVAX1 vectors using the method described already. The target fragment was detected by enzyme digestion (Fig. 1) and the sequence analysis showed that the inserted fragment in the pET-32a(+) and pVAX1 vectors was the 888 bp ORF of MIC2.

3.3. Expression and purification of recombinant MIC2 protein Recombinant plasmid conceiving cDNA fragment of MIC2 was expressed in vitro in E. coli BL21 (DE3). The recombinant protein was purified using the method described previously. High amount of recombinant MIC2 protein was obtained 6 h after the beginning of IPTG induction. The molecular weight of the expression fusion MIC2 protein was approximately 50.5 kDa on the SDS-PAGE gel (Fig. 2). Due to the fused protein of pET-32a(+) vector was about 20 kDa, the molecular weight of the recombinant protein of MIC2 was about 30.5 kDa, identical to the calculated value (30,604 Da).

3.4. Detection of the recombinant and native microneme proteins with western-blot The results of the immunoblot assay (Fig. 3) indicated that the recombinant MIC2 protein was recognized by immune sera of chickens infected with E. maxima, but the serum of normal chickens did not detect recombinant MIC2 protein. Western blot analysis also showed that ratanti-rMIC2 serum bound to a band of about 38 kDa in the somatic extract of E. maxima sporozoites (Fig. 3). The serum of unimmunized rat did not detect any protein of the somatic extract.

Fig. 3. Immunoblot for the recombinant MIC2 protein and native protein of MIC2. Lane M, standard protein molecular weight marker; Lane 1, recombinant MIC2 protein probed by serum of unimmunized chickens as primary antibody; Lane 2, recombinant MIC2 protein 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 ratanti-rEmMIC2 serum as primary antibody.

Fig. 4. Detection of expression of pVAX1-MIC2 in chicken muscle by RT-PCR. Lane M, DNA marker DL2000; Lane 1, non-injected muscle; Lane 2, pVAX1 plasmid injected muscle; Lane 3, pVAX1-MIC2 injected muscle.

Fig. 5. Detection of expression of pVAX1-MIC2 in chicken muscle by western blotting analysis. The samples were electro-transferred to nitrocellulose membranes and probed with rat-anti-rEmMIC2 serum as first antibody. Lane M, standard protein molecular weight marker; Lane 1, the muscle sample from chicken injected with pVAX1 plasmid; Lane 2, the muscle sample from chicken injected with pVAX1-MIC2.

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3.7. Proliferation responses of splenocytes As shown in Fig. 8, the splenocytes from both vaccinated groups of chickens displayed significantly greater proliferation compared with pVAX1 and PBS control (P b 0.05), however no significant differences were observed between the vaccinated groups. 3.8. Protective effects of MIC2 gene against E. maxima

Fig. 6. EmMIC2 specific IgG levels in chickens' serum. Each group of chickens was immunized with 100 μg of p VAX1–MIC2, 200 μg of rEmMIC2 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 was collected for determination of IgG levels weekly using ELISA method with respect to the absorbance at 450 nm, starting from the day of the first immunization, ending at the 4th week post the booster immunization. The concentrations of the IgG levels are expressed as mean ± S.D.

3.5. Identifications of the expression of MIC2 in vivo The results of RT-PCR indicated that the target fragment of MIC2 (approximately 888 bp) was detected from muscle RNA samples of chickens injected with pVAX1-MIC2 (Fig. 4). No specific bands were detected in non-injected control and pVAX1 plasmid control samples. Western blotting of the muscle of chickens injected with pVAX1MIC2 revealed a prominent band of 38 kDa, which indicated the expression of MIC2 gene (Fig. 5). No corresponding band was detected in the muscle of chickens injected with pVAX1.

3.6. IgG and cytokine levels in sera of immunized chickens The serum EmMIC2-specific IgG levels in chickens following vaccination with both rEmMIC2 and pVAX1-MIC2 are shown in Fig. 5. The anti-EmMIC2 antibody titers of both rEmMIC2 and pVAX1-MIC2 groups were significantly higher as compared to PBS and pVAX1 control (P b 0.05). Antibody titers increased highly in week 1 post the primary immunization and started to decrease at week 2 post-second immunization. Non-specific antibody was detected in PBS control and pVAX1 control groups throughout the experiment (Fig. 6). As depicted in Fig. 7, serum from chickens immunized with pVAX1-MIC2 and rEmMIC2 protein showed significantly high levels of IL-2 and IFN-γ (P b 0.05) compared to those of controls, within the same time, significantly high levels of IL-10 and IL-17 were also observed in chickens immunized with pVAX1-MIC2 and rEmMIC2 compared to those of negative controls. In the cases of the levels of TGF-β and IL-4, they are higher in vaccinated groups than those in unvaccinated groups (P b 0.05), though the elevation was modest, relatively speaking.

The efficacies of this immunization and challenge assay are presented in Table 1. No chicken died from coccidial challenge in any group in this study. Body weight gains were significantly reduced in positive and pVAX1 control groups compared with negative control (P b 0.05). Chickens immunized with rEmMIC2 and DNA vaccine pVAX1-MIC2 displayed significantly enhanced weight gains relative to chickens in challenged and pVAX1 control group (P b 0.05) (Fig. 9). The oocyst counts of immunized chickens were significantly lower than those of the positive and pVAX1 control groups (P b 0.05). Significant alleviations in jejunum lesions were observed in immunized chickens compared to that of challenged and pVAX1 control group (P b 0.05). Group of chickens immunized with DNA vaccine pVAX1MIC2 resulted in ACI more than 170, higher than that of recombinant EmMIC2 protein immunized group (Table 1). 4. Discussion Till now, a serious of microneme proteins of E. tenella have been cloned and their immunogenicity and functions have been tested [22–24]. However, few studies on E. maxima microneme proteins have been reported. In the current study, the immune protective effects against experimental E. maxima challenge of recombinant protein and DNA vaccine encoding EmMIC2 were evaluated. Our data showed that immunization with EmMIC2 could distinguishingly increase serum IgG antibody titers 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 EmMIC2 could provide ACIs of more than 165. These results showed that immunization with EmMIC2 could induce partial protection against E. maxima infection. In the present study, we demonstrated that the antibody titers in groups vaccinated with pVAX1-MIC2 and rEmMIC2 were significantly higher than those of PBS and pVAX1 control during weeks 1–5 post the first immunization (P b 0.05). The role of humoral immunity during coccidiosis was debatable with most evidence pointing to a minor function. However, recent studies have demonstrated that antibodies do play an important role in immunity against coccidiosis [25–27]. Our data are in high accordance with the recent results. IL-4 is a Th2-type cytokine and mainly regulates the antibody response [28]. In the present study, the level of IL-4 was significantly increased in the immunized groups. It strengthened the function of antibody response in the immunity against coccidiosis. It was reported that the responses of T-cells against Eimeria were partially controlled and regulated by cytokines [29]. 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 [30]. In previous research, the transcription levels of IFN-γ and IL-2 in chickens vaccinated with DNA vaccines were significantly increased and the chickens immunized with pVAX1-LDH-IL-2 and pVAX1-LDH-IFN-γ obtained higher

Fig. 7. Serum cytokines of chickens in different groups. Each group of chickens was immunized with 100 μg of p VAX1–MIC2, 200 μg of rEmMIC2 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 method 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 ± S.D. in pg/ml, IL-17 concentration (mean ± S.D.) in ng/ml. Bars with different letters are significantly different (P b 0.05). The concentration of (a) TGF-β; (b) IFN-γ; (c) IL-2; (d) IL-4; (e) IL-10; (f) IL-17.

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Fig. 8. Proliferation responses of splenic lymphocytes in chickens immunized with EmMIC2. Each group of chickens was immunized with 100 μg of p VAX1–MIC2, 200 μg of rEmMIC2 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, the spleens were removed and lymphocyte proliferation responses were determined by MTT method with respect to the absorbance at 570 nm. Bars with different letters are significantly different (P b 0.05).

oocyst decrease ratio and ACIs than chickens immunized with pVAX1LDH [31]. In the present study, we also observed that the concentrations of IFN-γ and IL-2 in the vaccinated chickens were at least 2.5 folds of that of the control groups. These data suggested that IFN-γ and IL-2 occupied a decisive position during immunization against coccidiosis. In this study, we also observed that the concentrations of IL-10, IL-17 and TGF-β in vaccinated chickens were significantly higher than those of the controls. However, these results seem to be controversial to previous studies. Th17-related cytokines displayed significantly higher level during oral experimental E. tenella infection, which indicated that IL-17 might facilitate the pathogenicity of E. tenella [32]. The expression of TGF-β mRNA was significantly increased in both the spleen and intestine following E. acervulina infection [33]. IL-10 might play a crucial role

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to block the development of strong IFN-γ-driven responses during infection of E. maxima [34]. Thus, the exact functions of IL-10, IL-17 and TGF-β in the infection and immune responses to vaccines against Eimeria need to be further investigated. It was reported that recombinant EtMIC1 antigen could induce IFN-γ response in vitro of spleenocytes from E. tenella infected birds [35]. This indicted that recombinant EtMIC1 protein might induce T-cell response in birds, which is crucial for conferring protective immunity against Eimeria infection. In the present study, our data showed that the splenocytes from chickens of both groups vaccinated with pVAX1-MIC2 and rEmMIC2 displayed significantly greater proliferation compared with controls (P b 0.05). The IFN-γ was also enhanced in our study. These results showed that immunization with EmMIC2 gene could induce cell-based immune responses. In the present research, MTT assay was employed to investigate the effects of EmMIC2 proteins on the functions of lymphocytes in vivo. Con A is a plant mitogen and is known for its ability to stimulate T cells 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 [36]. In this study, the splenocytes from both vaccinated groups of chickens displayed significantly greater proliferation compared with pVAX1 and PBS control (P b 0.05). It indicated that EmMIC2 formulated as both subunit and DNA vaccines could enhance the proliferation of T cells to Con A stimulation and thus increase the functional potential of the cells. The challenge experiment results showed that both the recombinant protein and the DNA vaccine could significantly alleviate jejunum lesions and increase oocyst decrease ratio compared to the positive control. Similar results have also been revealed that vaccination with the Gam82 recombinant protein could induce protective intestinal immunity against E. maxima, as revealed by decreased oocyst shedding and reduced gut pathology, which was associated with increased humoral and cell-mediated immune responses [37]. These results suggested that host immunity might play an important role in impeding host transmission of Eimeria infection. In the present study, the ACIs of chickens immunized with DNA vaccine pVAX1-MIC2 and rEmMIC2 were 170.2 and 165.8, respectively, suggesting that partial protection was induced. The weight gain has proven to be the most useful criterion for evaluating the efficacy of anti-coccidial drugs during the acute phase of infection [7, 38]. In the current work, the weight gains of the vaccinated birds were significantly higher than that of the control birds, but still significantly lower than that of the unchallenged controls. This indicated that the protection of pVAX1-MIC2 and the corresponding recombinant antigen should be further improved. In the current work, the anti-rEmMIC2 sera recognized a band of about 38 kDa in the somatic extract of E. maxima sporozoites. These results suggested that the native MIC2 was about 38 kDa, slightly larger than the calculated size. Using the prediction server, 2 O-glycosylation sites and 23 phosphorylation sites were detected. The glycosylation and phosphorylation might enlarge the size of the native MIC2 compared to the calculated one (30.6 kDa). In conclusion, our data in the present study demonstrated that immunization with EmMIIC2 could induce both humoral and cellmediated immune responses to E. maxima and resulted in partial protection against challenge in chickens. This indicated that EmMIC2 might be an effective antigen candidate for the development of a new vaccine against E. maxima.

Acknowledgments

Fig. 9. The relative ratio of body weight gain (percentage of negative control). Bars with different letters are significantly different (P b 0.05).

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

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