Co-expression of EtMic2 protein and chicken interleukin-18 for DNA vaccine against chicken coccidiosis.

ARTICLE IN PRESS Research in Veterinary Science ■■ (2014) ■■–■■

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Research in Veterinary Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / r v s c

Co-expression of EtMic2 protein and chicken interleukin-18 for DNA vaccine against chicken coccidiosis Wenyan Shi a,b,1, Qing Liu a,b,1, Jie Zhang a,b, Jingjing Sun a,b, Xiyue Jiang Fangkun Wang a,b, Yihong Xiao c, Hongmei Li a,b,**, Xiaomin Zhao a,b,*

a,b

, Jing Geng

a,b

,

a

Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, 61 Daizong Street, Taian City, Shandong Province 271018, China b Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Agricultural University, 61 Daizong Street, Taian City, Shandong Province 271018, China c Department of Basic Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, 61 Daizong Street, Taian City, Shandong Province 271018, China

A R T I C L E

I N F O

Article history: Received 21 December 2013 Accepted 3 May 2014 Keywords: EtMIC2 ChIL-18 Co-expression DNA vaccine

A B S T R A C T

In the present study, a naked EtMIC2 DNA vaccine, a ChIL-18 expression vector and a EtMIC2 and ChIL-18 co-expression DNA vaccine were constructed and their protective efficacies against homologous challenge were compared and evaluated by examining the body weight gain, oocyst shedding, cecal lesion, ACI as well as specific anti-EtMic2 antibody level, the proliferation ability and percentages of CD4+ and CD8+ of splenocytes. The results showed the naked EtMIC2 DNA vaccine could increase the weight gain and decrease the oocyst shedding, but could not alleviate the cecal lesion of immunized chickens compared to unimmunized chickens. Chickens immunized with the co-expression vector pVAX1-MIC2-IL18 exhibited much improved immune protection against challenge compared to chickens immunized with naked EtMIC2 DNA vaccine, or with naked EtMIC2 DNA vaccine and ChIL-18 expression vector applied separately. These results suggest that the co-expression of ChIL-18 with EtMic2 together could significantly improve the immune protection of the EtMic2 protein. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction The chicken coccidiosis, a protozoan parasitic disease caused by intracellular apicomplexan parasite Eimeria species (spp), leads to considerable economic losses to the avian industry worldwide. The main control strategies against avian coccidiosis are the use of anticoccidial drugs and live oocysts vaccine (Shirley et al., 2005). However, the use of anti-coccidial drugs has been greatly restricted with the continual emergence of drug resistance, the expensive cost with developing new drugs, the drug residues, and the public concern of safety in animal foodstuff. Meanwhile, the use of alive oocyst vaccine has some drawbacks including the pathogenicity, high production expenses and atavistic possibility of coccidia (Sharman et al., 2010; Vermeulen, 1998). Therefore, novel approaches are urgently needed to control the chicken coccidiosis. With the rapid progress of the molecular biology, the DNA vaccine

1 Authors contributed equally to this work. * Corresponding author. Tel.: +86 0538 8249921; fax: +86 538 8249921. E-mail address: [email protected] (X. Zhao). ** Corresponding author. Tel.: +86 0538 8249921; fax: +86 538 8249921. E-mail address: [email protected] (H. Li).

development has attracted much attention for its unique advantages, including the ability to induce a wider range of immune response types, with no risk for infection, ease of development and production, stability for storage and shipping and cost-effectiveness (Lillehoj et al., 2000; Vermeulen, 1998; Wu et al., 2005). Although work on DNA vaccines against Eimeria spp. is still in its early stages, it has shown a bright future. Several promising vaccine candidates containing immunogenic proteins of Eimeria spp. have been described. It was reported that vaccination with recombinant 3-IE, SO7, TA4, EtMIC2, and 5401 proteins of Eimeria spp. induced protective intestinal immunity against coccidiosis (Ding et al., 2004; Du and Wang, 2005; Kopko et al., 2000; Wu et al., 2004). Several researchers also tried in ovo DNA vaccinations and revealed that ovo DNA vaccination might provide the earlier onset of immunity against homologous challenge (Ding et al., 2005; Haygreen et al., 2005, 2006). Several studies reported that the efficiency of DNA vaccination could be significantly enhanced by cytokines as adjuvants (Lillehoj and Choi, 1998; Ma et al., 2011; Shah et al., 2010, 2011; Song et al., 2013). Recently, studies revealed that the cytokine IL-18 could enhance the protection efficacy of DNA immunization and host immune responses (Degen et al., 2005). IL-18 can induce the Th1-mediated cellular immunity and Th2-mediated humoral immunity, enhance the activity of NK cell and CTL cell, promote immune cells express FasL-

http://dx.doi.org/10.1016/j.rvsc.2014.05.001 0034-5288/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Wenyan Shi, et al., Co-expression of EtMic2 protein and chicken interleukin-18 for DNA vaccine against chicken coccidiosis, Research in Veterinary Science (2014), doi: 10.1016/j.rvsc.2014.05.001

ARTICLE IN PRESS W. Shi et al./Research in Veterinary Science ■■ (2014) ■■–■■

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mediated cytotoxicity, and thus plays an important role in host defense (Kinoshita et al., 2013; Wong et al., 2013). Yan et al. (2012) reported that chicken IL-18 (ChIL-18) could significantly improve the humoral and cellular immunity of host when applied together with a DNA vaccine encoding a perforin-like protein 1 (Plp1)/microneme protein 6 (Mic6) of Toxoplasma gondii compared with naked pIRESneo/MIC6/PLP1 immunization. The E. tenella microneme-2 protein (EtMic2) is secreted from the microneme and involved in coccidian invasion (Kawazoe et al., 1992). Previous studies showed that the EtMic2 protein has good immunogenicity and could provoke partial protection of the chicken from E. tenella infections and might be a good candidate for use in vaccine development (Ding et al., 2005; Lillehoj et al., 2005; Sathish et al., 2011). In the present study, we constructed a naked EtMIC2 DNA vaccine, a ChIL-18 expression vector and a co-expression DNA vaccine of EtMIC2 and ChIL-18. Their protective efficacies against homologous challenge were compared and evaluated.

from pET-ChIL-18 plasmid (kindly provided by Dr. Jingdong Hu, Shandong Agricultural University, China) using primers of ChIL-18-F (5′-CCCGAATTCACGATGGCCTTTTGTAAGGAT-3′) and ChIL-18-R (5′TTTCTCGAGTCAATGATGATGATGATGATGTAGGTTGTGCCTTTC-3′). The PCR products were digested with enzymes EcoR I and Xho I and cloned into the pVAX1.0 vector which was digested with the same enzymes to generate plasmid pVAX1-IL-18. To construct pVAX1MIC2-IL18 for co-expression of EtMic2 and ChIL-18 proteins, the primers of MIC2-IL18-F (5′-TTTGAATTCGGTGGAGGAGGCTCTGC CTTTTGTAAGGATAAAACTATC-3′) and ChIL-18-R were used to amplify the entire coding region of the ChIL-18 gene. The forward primer MIC2-IL18-F was designed to contain the sequence encoding five amino acids (GGGGS) which serve as a linker between the EtMic2 and ChIL-18 proteins. The PCR products were digested with EcoR I and Xho I and cloned into plasmid pVAX1-MIC2 at EcoR I/Xho I sites to construct pVAX1-MIC2-IL18. The three recombinant plasmids were confirmed by PCR amplification, endonuclease cleavage and sequencing analysis.

2. Materials and methods 2.3. Expression analysis of the recombinant plasmids in vitro and in vivo

2.1. Parasites and experimental chickens The wild type E. tenella strain SD-01 was stored in our laboratory. Sporulated oocysts were stored in 2.5% potassium dichromate at 4 °C and propagated in 3 week old chickens every 6 months as previously described (Fetterer and Barfield, 2003). The sporulated oocysts for the experiments were purified from newly infected chickens. One day old male Hy-Line Variety Brown layer chickens (Dongyue poultry, Taian, China) were reared in isolated cabinets under coccidian-free conditions and provided with coccidiostat-free feed and water ad libitum. Chickens were randomly divided into 7 groups of 20 birds each (Table 1). 2.2. Construction of recombinant plasmids pVAX1-MIC2, pVAX1-IL18 and pVAX1-MIC2-IL-18 E. coli strain TOP10 (Invitrogen, USA) was used for recombinant DNA manipulation. E. coli was grown in Luria-Bertani (LB) medium (0.5% yeast extract, 1% tryptone and 1% NaCl) at 37 °C with shaking at 220 rpm. The E. coli transformants were selected on LB containing kanamycin (100 μg/ml) plates cultured at 37 °C. The plasmid pVAX1.0 was used as the eukaryotic expression vector to generate the recombinant plasmids. The entire coding region of the EtMIC2 gene was PCR amplified from a EtMIC2 containing plasmid stored in our laboratory using primers of EtMIC2-F (5′-TTTGGATCCAACATGGTCCCAGGCGAAGATAGCTTC-3′) and EtMIC2-R (5′-TTT GAATTCGGATGACTGTTGAGTGTCACT-3′) using pfu polymerase. The PCR products were digested with BamH I and EcoR I and agarose gel purified using Easy Pure Quick Gel Extraction Kit (TransGen, China). The purified PCR products were ligated into pVAX1.0 vector which was digested with the same enzymes to generate plasmid pVAX1-MIC2. Similarly, ChIL-18 gene was amplified

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

Immunogen (μg)

Immunization (day)

Challenge (day)

I II III IV V VI VII

/ / pVAX1.0 vector (100) pVAX1-MIC2 (100) pVAX1-IL-18 (100) pVAX1-MIC2-IL-18 (100) pVAX1-MIC2 (100) mixed pVAX1-IL-18 (100)

/ / 7, 14, 21 7, 14, 21 7, 14, 21 7, 14, 21 7, 14, 21

/ 28 28 28 28 28 28

To test if the constructed recombinant plasmids work, the expression analysis of the plasmids was performed both in vitro and in vivo. For expression analysis in vitro, recombinant plasmids pVAX1MIC2, pVAX1-IL-18 and pVAX1-MIC2-IL-18 were transfected into 293T cells with lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer’s instructions. The indirect immunofluorescence analysis (IFA) and Western-blot methods were employed to detect the expression of the recombinant plasmids. Twentyfour hours after transfection, cells were fixed with fixative (acetone: ethanol = 3:2) for 8 min. After washing with PBS for three times, the cells were incubated with anti-EtMic2 polyclonal antibody (1:1000, stored in our laboratory) or ChIL-18 polyclonal antibody (1:1000, raised in our laboratory) at 37 °C for 1 h. After washing with PBS for three times, the plates were incubated with FITC-labeled goat antimouse IgG antibody (1:75; CWBIO, China) at 37 °C for 1 h. After washing, fluorescences were examined under a fluorescence microscope (Nikon, Japan). The 293T cells transfected with vector pVAX1.0 were served as the negative control. For Western-blot analysis, cells transfected with the recombinant plasmids were washed with PBS and then lysed using cell lyses buffer (Beyotime, China). The cell lysates were centrifuged at 12,000 rpm for 2 min at 4 °C. The cell proteins in the supernatant were separated by 10% SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) with the condition of 150 mA, 2 h. Then the PVDF membrane was blocked with 5% skimmed milk powder (CWBIO, China) overnight at 4 °C. After washing with TBST three times, the PVDF membranes were incubated with anti-Mic2 antibody (1:500) and anti-His Tag monoclonal antibody (1:2000; TIANGEN, China) respectively at 37 °C for 1 h. After washing with TBST three times, the PVDF membranes were incubated with HRP-labeled goat anti-mouse IgG antibody (1:75; CWBIO, China) at 37 °C for 1 h. After washing, DAB reagent (TIANGEN, China) was used for color development. For expression analysis in vivo, 18-day-old Kunming mice (China Biologic Products, Inc) were randomly divided into five groups with five mice in each group. Four groups were immunized with 50 μg plasmids of pVAX1-MIC2, pVAX1-IL-18, pVAX1-MIC2-IL-18 and pVAX1.0 respectively. One group was severed as blank control. The mice were immunized three times by leg intramuscular injection at 18, 32 and 46 days respectively. Both anti-EtMic2 antibody and anti-ChIL18 antibody were measured from the blood sample at 46 and 60 days with ELISA as described by Sun et al. (2014).



2.4. Immunization and parasite-challenge infection At 7, 14, 21 days of age, chickens of the experimental groups were immunized with 100 μg DNA vaccines according to the experimental design as described in Table 1 by leg intramuscular injection. To help the vaccine plasmids express, 200 μl 25% sucrose solution was injected and kneaded the leg lightly before immunization injections. All chickens except the unchallenged group (group I) were challenged orally with 6000 sporulated oocysts of E. tenella at 28 days. 2.5. Evaluation of protective efficacy The protective efficacy for each experimental group was evaluated on the basis of survival rate, body weight gain, relative growth rate, cecal lesion score, oocyst output and anti-coccidial index (ACI). The survival rate was determined by the number of surviving chickens divided by the number of initial chickens in each group. The body weight gain was determined by the body weight of the chickens at the end of the experiments subtracting the body weight at the time of challenge. The growth rate was calculated as the body weight gain divided by the body weight at the time of challenge. The relative growth rate was calculated as growth rate of vaccinated group divided by growth rate of group I. At 7 day post challenge, the cecal lesion score of chickens from each group was recorded using a numerical scale from 0 (normal) to 4 (severe) following the method described by Johnson and Reid (1970). The lesion score was determined by three independent observers. The oocyst numbers per gram of the cecal content (OPG) were counted microscopically from 1 g of the cecal content using McMaster’s counting technique. The oocyst decrease rate was calculated as described by Rose and Mockett (1983) as follows: (the mean number of oocysts from the challenged control chickens − the mean number of oocysts from vaccinated chickens)/ the mean number of oocysts from the challenged control chickens × 100%. ACI was calculated following the method described by Chapman and Shirley (1989): (survival rate + relative growth rate) − (lesion value + oocyst value). 2.6. Specific antibody measurement Blood samples were collected from three chickens chosen randomly in each group at days 7, 14, 21, 28 and 35. The serum prepared from the blood by low speed centrifugation was stored at −20 °C until further analysis. The level of the specific anti-EtMic2 antibody was examined by ELISA. Briefly, 96-well microtiter plates were coated with EtMic2 protein (200 ng/well) and incubated overnight at 4 °C. The coated plates were washed with PBS containing 0.05% Tween-20 (PBST), and blocked with 5% non-fat milk in PBST for 1 h at 37 °C. After washing with PBST, 100 μl diluted serum samples (1:10) was added into each well and incubated for 1 h at 37 °C. The plates were washed twice with PBST. Then 100 μl of the second antibody (HRP-conjugated rabbit anti-chicken IgM or IgG, Bioss, China) with 1:1000 dilution was added to each well and the plates were incubated for 1 h at 37 °C. Optical density values at 450 nm (OD450) were measured using an automated microplate reader (Biotek, USA). All samples were analyzed in triplicates. 2.7. Splenocytes analysis For the splenocyte proliferation analysis, the spleens from birds of the experimental and control groups were removed from six chickens of each group at 35 days as described by Sasai et al. (2000). Splenocytes were isolated using chicken lymphocytes separation solution (Solarbio, China) according to the manufacturer’s protocol. Single-cell clones from spleens were prepared as described (Sasai et al., 2000). The 100 μl splenocytes (5 × 105/ml) from sing-cell clones prepared earlier were added to each well of a 96-well plates and

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100 μl RPMI 1640 medium were used as control. To each well (except the controls), 10 μl 0.5 mg/ml ConcanavalinA (ConA, Sigma, USA) was added and mixed well. The plates were incubated at 37 °C for 66 h in a 5% CO2 atmosphere. Then 70 μl supernatant from each well was abandoned and 10 μl 0.5% MTT (Sigma, USA) was added. After 4 h incubation, 100 μl DMF was added to each well. Continued to incubate for 2 h with shaking and then the OD value was read at 490 nm in a microplate reader. The cells without ConA stimulation were used as controls. The experiment was repeated four times. For the analysis of the subsets of CD4+ and CD8+ splenocytes, the splenocytes from sing-cell clones prepared earlier were incubated with R-PE-conjugated mouse anti-chickens CD8 α antibody (0.1 mg/ml) (Abcam, USA) and FITC- conjugated mouse antichickens CD4 antibody (0.5 mg/ml) (Abcam, USA) for 20 min at 4 °C in dark, and then detected by flow cytometry (Guava, USA) for evaluation of the percentages of CD4+ and CD8+ cells. 2.8. Statistical analysis The data were statistically analyzed using software SPSS 17.0 (SPSS Inc., Chicago, IL) for variance and Duncan’s multiple ranges. P < 0.05 was considered statistically significant. 3. Results 3.1. Construction and expression of the recombinant plasmids To evaluate efficacy of the ChIL-18 as immune adjuvant of the EtMic2 DNA vaccine in different combination ways, the recombinant plasmids pVAX1-MIC2, pVAX1-IL-18 and pVAX1-MIC2-IL-18 were constructed as described in ‘Materials and methods’. The resultant plasmids were verified by PCR, restriction enzyme digestion and double strand sequencing (data not shown). The IFA results showed that the 293T cells transfected with recombinant plasmids pVAX1-MIC2 and pVAX1-MIC2-IL-18 were fluorescent labeled positive using anti-EtMic2 polyclonal antibody (Fig. 1: B1, B2, D1, D2), and the 293T cells transfected with recombinant plasmids pVAX1-IL-18 and pVAX1-MIC2-IL-18 were fluorescent labeled positive using anti-ChIL-18 polyclonal antibody (Fig. 1: C1, C2, E1, E2), while no fluorescence was detected in cells transfected with pVAX1.0 vector (Fig. 1: A1, A2). The Western-blot analysis showed that the 293T cell transfection with pVAX1MIC2, pVAX1-IL-18 and pVAX1-MIC2-IL-18 plasmids resulted in the expression of the EtMic2, ChIL-18 proteins, and EtMic2-ChIL-18 fusion proteins with the sizes of 45, 19 and 65 kD respectively, which are similar to the predicted size. The protein EtMic2 and fusion protein EtMic2-ChIL-18 were specifically probed by anti-EtMic2 antibody (Fig. 2A). The protein ChIL-18 and fusion protein EtMic2-ChIL-18 were specifically probed by anti-His Tag monoclonal antibody (Fig. 2B).Whereas no band was detected from cells transfected with pVAX1.0 vector and normal cells. For the expression analysis of the plasmids in vivo, the immunizations of the plasmids in animals were performed. In order to avoid the influence of the chicken native ChIL-18 protein on the analysis, the mice were used as the test animals. The results showed that the specific anti-EtMic2 and anti-ChIL-18 antibodies in the immunized group were detected in a significantly higher level than that in control groups at both 46 and 60 days (Fig. 3). The results provided evidence that EtMIC2 gene and ChIL-18 gene in recombinant plasmids were successfully expressed in vivo. 3.2. Protective efficacy of vaccination against E. tenella challenge The protective efficacy of the recombinant plasmids was evaluated based on the body weight gain, relative growth rate, cecal lesion score, OPG, oocyst decrease rate, and ACI. The results revealed that


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Fig. 1. IFA detection of proteins EtMic2 and ChIL-18 expressed in 293T cells transfected with recombinant plasmids. (A) pVAX1.0 transfected cells were stained with antiEtMic2 antibody and FITC-conjugated goat anti-mouse IgG. (A1) Under normal white light. (A2) Under the FITC filter. (B) pVAX1-MIC2 transfected cells were stained with anti-EtMic2 antibody and FITC-conjugated goat anti-mouse IgG. (B1) Under normal white light. (B2) Under the FITC filter. (C) pVAX1-IL-18 transfected cells were stained with anti-ChIL-18 antibody and FITC-conjugated goat anti-mouse IgG. (C1) Under normal white light. (C2) Under the FITC filter. (D) pVAX1-MIC2-IL-18 transfected cells were stained with anti-EtMic2 antibody and FITC-conjugated goat anti-mouse IgG. (D1) Under normal white light. (D2) Under the FITC filter. (E) pVAX1-MIC2-IL-18 transfected cells were stained with anti-ChIL-18 antibody and FITC-conjugated goat anti-mouse IgG. (E1) Under normal white light. (E2) Under the FITC filter.



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of 77.84 ± 9.46, which is significantly higher than that in groups IV, V, and VII (Table 2). The cecal lesion score was measured using a numerical scale from 0 (normal) to 4 (severe). The cecal lesion scores in groups IV, V, VII were observed reduced compared to the challenged control groups (II–III), but the difference was not statistically significant. The average cecal lesion score in group VI was significantly decreased (P < 0.01) than that of the challenged control groups and other immunized group chickens (Table 2). The oocyst shedding of chickens in all immunized groups (IV– VII) was greatly reduced compared to controls (Table 2). 3.3. Serum specific antibody levels

Fig. 2. Western blot analysis of proteins EtMic2 and ChIL-18 expressed in 293T cells transfected with recombinant plasmids. M: protein marker; lane 1: yeast expressed EtMic2 protein; lane 2: protein mixture of the pVAX1-MIC2-IL-18 transfected 293T cell lysates; lane 3: protein mixture of the pVAX1-IL-18 transfected 293T cell lysates; lane 4: protein mixture of the pVAX1-MIC2 transfected 293T cell lysates; lane 5: protein mixture of the pVAX1.0 vector transfected 293T cell lysates; lane 6: protein mixture of the uninfected 293T cell lysates used as negative control. (A) AntiEtMic2 antibody and HRP-labeled goat anti-mouse IgG antibody were used for analysis. (B) Anti-His Tag monoclonal antibody and HRP-labeled goat anti-mouse IgG antibody were used for analysis.

In order to evaluate the humoral immune response, the serum specific anti-EtMic2 antibody was determined using ELISA. No obvious difference of the serum specific anti-EtMic2 antibody among all groups was observed on days 7, 14 and 21. The difference was not significant in all groups on day 28. On day 35 (14 days after last immunization), the antibody levels were significantly increased in chickens of all challenged groups and a very significant difference (P < 0.01) was observed in group VI compared to other groups (Fig. 4). 3.4. Proliferation and subsets of splenocytes

the chickens in non-immunized-challenged control group (group II) and the chickens immunized with pVAX1.0 (group III) exhibited significantly (P < 0.01) reduced weight gain, higher average cecal lesion score, and higher OPG as compared to the unchallenged control group chickens (group I) and compared to all vaccinated chickens (Table 2). No chicken died from E. tenella challenge in any group. All the vaccinated chickens significantly improved the average weight gain compared to groups II and III. Among the vaccinated groups, the chickens in group VI (immunized with pVAX1-MIC2IL-18) received the best weight gain with the average weight gain

To evaluate the effects of the immunization with various DNA vaccines on the immune functions, the splenocyte proliferation ability and the subsets of CD4+ and CD8+ splenocytes were measured. The splenocyte proliferation levels with ConA or without ConA stimulation were detected using ELISA. The splenocyte proliferation values without ConA stimulation from all challenged groups showed no significant difference (P > 0.05) and were significantly higher than that in group I (P < 0.05). After stimulation with ConA, the splenocytes exhibited increased proliferation ability in all groups. The spleno-

Fig. 3. The anti-EtMic2 antibody and anti-ChIL-18 antibody levels of mice immunized with recombinant plasmids. Serum was collected and analyzed by ELISA. Each bar represents the mean ± SD values (n = 5) and each sample was analyzed in triplicate. Bars not sharing the same letters were significantly different according to Duncan’s multiple range (P < 0.05).

Table 2 Protective efficacy of vaccination against E. tenella challenge. Group

BWG (g)


90.00 ± 7.57 63.37 ± 8.31d 64.01 ± 15.0d 72.28 ± 3.65b,c 68.89 ± 4.35c,d 77.84 ± 9.46b 74.46 ± 6.47c a

RGR (%)

LS

100 70.25 70.96 80.27 75.86 86.67 82.55

0.00 ± 0.00 3.40 ± 0.843c 3.33 ± 0.866c 2.75 ± 1.22c 2.92 ± 1.08c 1.45 ± 1.37b 2.50 ± 1.09c a

OPG (×106)

ODR (%)

ACI

0 3.67 2.89 1.24 1.85 0.43 0.96

100 0 21.25 66.21 49.59 88.28 73.84

200 126.25 127.66 152.77 145.66 171.17 156.55

a, b, c, d: different letter means different statistical significance. BWG, body weight gain; RGR, relative growth rate; LS, lesion score; OPG, oocyst numbers per gram of the cecal content; ODR, oocyst decrease rate; ACI, anti-coccidial index.



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Fig. 4. The anti-EtMic2 antibody level of chicken immunized with recombinant plasmids. Serum was collected and analyzed by ELISA. Each bar represents the mean ± SD values (n = 3) and each sample was analyzed in triplicates. Bars not sharing the same letters were significantly different according to Duncan’s multiple range (P < 0.05).

cyte proliferation values with ConA stimulation of chickens in all immunized groups (IV–VII) were significantly higher compared to controls, and the value in groups VI was significantly higher (P < 0.05) than that of all other groups except group IV (Table 3). There was significant increase of CD4+ splenocytes in group VI compared to that in other groups (P < 0.01). The CD4+ splenocyte percentage in group VII was significantly higher than those in other groups except group I (P < 0.01). The percentage of CD8+ splenocytes in group VI was not different from group I, and significantly (P < 0.01) higher than that in other groups (Table 3). 4. Discussion Chicken coccidiosis causes huge economic losses in poultry industry. As the emergence of drug resistance of Eimeria species and the environmental pollution of the live oocyst vaccine, the development of new vaccine types against Eimeria species has draw more attentions of parasitologists worldwide (Shams, 2005; Vermeulen et al., 2001). Since Kopko et al. (2000) first constructed a DNA vaccine using plasmid pcDNA3 by expressing the SO7 gene of E. tenella, several studies on DNA vaccines against Eimeria species have been published later on (Du and Wang, 2005; Wu et al., 2004). Ding et al. (2005) conducted in ovo vaccination embryonated eggs with pcDNAEtMIC2 and the birds hatched from the vaccinated eggs developed partial protective immunity against infection by E. tenella as assessed by significantly increased body weight gain and decreased fecal oocyst shedding compared with non-vaccinated controls. Although DNA vaccines against Eimeria species reported could induce relatively good protective immunity, it seemed that the protection using naked DNA vaccination against challenge was variable and not completed (Shirley et al., 2005). Recent studies reported that cytokines as adjuvant with antigens could enhance immune responses against challenge. It is documented that DNA vaccination

of chickens in combination with cytokines such as IL-2, IL-15, and IFN-γ enhanced protective intestinal immunity against coccidiosis (Lillehoj and Choi, 1998; Lillehoj et al., 2005; Shah et al., 2010). Shah et al. (2010) reported that the ACI of pVAX-1-cSZ-2 immunization group was 154.6 and in the group immunized with pVAX-1-cSZ-2chIL-2 showed a higher ACI (182.2). The similar results were obtained by Song et al. (2013) who constructed DNA vaccine with SO7 and used chIL-2 as adjuvant to immune chickens against E. tenella. In the present study, EtMIC2 gene as vaccine target, ChIL-18 as immune enhancement factor and pVAX1.0 as the eukaryotic expression vector were used to develop recombinant plasmids as DNA vaccines against chicken coccidiosis. Protective efficacy and immunogenicity of the plasmids expressing EtMic2 alone (pVAX1MIC2), co-expressing EtMic2 and ChIL-18 (pVAX1-MIC2-IL-18), plasmids expressing EtMic2 and ChIL-18 separately (pVAX1MIC2 + pVAX1-IL-18) were evaluated. The results showed that chickens immunized with pVAX1-MIC2 alone could alleviate the disease by significantly increasing the average weight gain and decreasing the oocyst shedding compared to unimmunized chickens and immunized chickens with vector; the average cecal lesion in this group was observed slightly lighter than that of unimmunized chickens and immunized chickens with vector, but the difference was not significant (Table 2). The ACI of this group was accessed as about 153 which suggests that the protection against challenge was not good enough. Chickens immunized with co-expression vector pVAX1MIC2-IL-18 exhibited much improved immune protection against challenge. The average weight gain was significantly higher, and the oocyst shedding and average cecal lesion score were significantly lower than that of challenged control groups and other vaccinated groups. The ACI reached 171 which suggest a good protection against challenge. These results suggest that the co-expression of ChIL-18 with EtMic2 together could significantly improve the immune protection of the naked EtMic2 vaccine.

Table 3 The percentages of CD4+, CD8+ splenocytes and splenocytes proliferation. Group


Percentages of CD4+/CD8+ splenocytes

Splenocytes proliferation

CD4+ (%)

CD8+ (%)

No ConA stimulated

ConA stimulated

15.496 ± 2.053b 12.992 ± 0.8407a 13.152 ± 1.431a 13.24 ± 1.681a 13.336 ± 0.9839a 22.79 ± 1.341c 17.244 ± 0.7921b

23.88 ± 3.148c 14.18 ± 0.8157a 15.287 ± 1.271a 17.8 ± 0.8735b 17.373 ± 1.046b 23.523 ± 1.165c 18.155 ± 0.5118b

0.185 ± 0.0067b 0.266 ± 0.0305a 0.248 ± 0.0172a 0.260 ± 0.0107a 0.250 ± 0.0177a 0.267 ± 0.0238a 0.253 ± 0.0106a

0.215 ± 0.0074d 0.327 ± 0.0225c 0.314 ± 0.0173c 0.360 ± 0.0189a,b 0.348 ± 0.0144b 0.379 ± 0.0209a 0.343 ± 0.0293b

a, b, c, d: different letters mean different statistical significance.



It is worth noting that although the co-expression of ChIL-18 could significantly enhance the immune protection of the naked EtMic2 vaccine against challenge, the vaccination of the expression vector of ChIL-18 (pVAX1-IL-18) applied separately with the expression vector of EtMic2 (pVAX1-MIC2) could not improve the protection of the naked EtMic2 vaccine, which suggests that the adjuvant function of the ChIL-18 is condition dependent. It was believed from early studies that cellular immunity plays the most important role in protection against Eimeria spp, whereas humoral immunity plays a very minor role in resistance against infection (Rose and Long, 1971; Sathish et al., 2011). However, recent several reports suggested that the antibodies raised by live immunization or against purified stage-specific Eimeria antigens have ability to inhibit parasite development both in vitro and in vivo, which revealed the role of antibody in protection against coccidiosis (Wallach, 2010). In the present study, we measured the antiEtMic2 specific antibody levels, the proliferation ability and percentages of CD4+ and CD8+ of splenocytes in all the groups to evaluate the roles of the humoral and cellular immunities in protection against Eimeria spp infection. The antibody levels were significantly increased in chickens of all challenged groups on day 35 (14 days after last immunization). A significant increase (P < 0.01) was observed in chickens of group VI (immunized with co-expression plasmid pVAX1-MIC2-IL-18) compared to other groups (Fig. 4). These results are congruent with the data of body weight gain, cecal lesion score, oocyst decrease rate, and ACI, which suggests that the specific antibody may play roles in protection against challenge. In conclusion, the results demonstrated that the naked EtMIC2 DNA vaccine could alleviate the disease by significantly increasing the weight gain and decreasing the oocyst shedding compared to unimmunized chickens. Co-expression of ChIL-18 with EtMic2 together could significantly improve the immune protection of chickens against challenge compared to chickens immunized with naked EtMic2 vaccine.

Acknowledgments This work was supported by a grant from Shandong Province Science and Technology Development Program to XZ (No. 2013GNC11017) and a grant from the National Natural Science Foundation of China to XZ (No. 31172314).

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