Identification and immunogenicity of microneme protein 2 (EbMIC2) of Eimeria brunetti

Experimental Parasitology 162 (2016) 7e17

Contents lists available at ScienceDirect

Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr

Full length article

Identification and immunogenicity of microneme protein 2 (EbMIC2) of Eimeria brunetti Tran Duc Hoan, Zhenchao Zhang, Jingwei Huang, Ruofeng Yan, Xiaokai Song, Lixin Xu, Xiangrui Li* College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu, PR China

h i g h l i g h t s

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


The clone had no significant differences with the EbMIC2 deposited in the GenBank.
The expressed recombinant protein could have high immunogenicity.
Recombinant EbMIC2 could protection against homologous challenge in chicken.
Serum from challenged chickens showed significantly compared with control groups.
Parameters of challenged groups showed significantly compared with unchallenged.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 January 2015 Received in revised form 8 December 2015 Accepted 22 December 2015 Available online 30 December 2015

There have been only a few antigen genes of Eimeria brunetti reported up to now. In this study, the gene encoding the microneme protein 2 (EbMIC2) was isolated from oocysts of E. brunetti by RT-PCR and the immunogenicity of recombinant EbMIC2 was observed. The EbMIC2 was cloned into vector pMD19-T for sequencing. The sequence was compared with the published EbMIC2 gene from GenBank revealed homology of the nucleotide sequence and amino acids sequence were 99.43 and 98.63%, respectively. The correct recombinant pMD-EbMIC2 plasmid was inserted into the pET-28a (þ) expressing vector and transformed into competent Escherichia coli BL21 cells for expression. The expressed product was analyzed using SDS-PAGE and Western-blot. The results indicated that the recombinant EbMIC2 protein was recognized strongly by serum from naturally infected chicken with E. brunetti. Rat rcEbMIC2 antisera bound to bands of about 36 kDa in the somatic extract of E. brunetti sporozoites. The recombinant plasmid pVAX1-EbMIC2 was constructed and then the efficacies of recombinant plasmid and recombinant protein were evaluated. The results of IgG antibody level and cytokines concentration suggested that recombinant EbMIC2 could increase the IgG antibody level and induce the expressions of cytokines. Animal challenge experiments demonstrated that the recombinant EbMIC2 protein and recombinant plasmid pVAX1-EbMIC2 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens and presented high anti-coccidial index. All results suggested that EbMIC2 could become an effective candidate for the development of new vaccine against E. brunetti infection. © 2015 Elsevier Inc. All rights reserved.

Keywords: Eimeria brunetti Microneme-2 Recombinant plasmid Recombinant protein Chicken

* Corresponding author. E-mail address: [email protected] (X. Li). http://dx.doi.org/10.1016/j.exppara.2015.12.015 0014-4894/© 2015 Elsevier Inc. All rights reserved.

8

T.D. Hoan et al. / Experimental Parasitology 162 (2016) 7e17

1. Introduction Coccidiosis is an intestinal disease caused by protozoan parasites of the genus Eimeria (Anders and Jørgen, 2005; Donal and Elizabeth, 2007). It can easily occur in intensive farm and brings a mortality of 50e90% for poultry (Morris and Gasser, 2006). The present control strategy against chicken coccidiosis mainly employs the use of anti-coccidial drugs. Due to rapid emergence of drug resistant strains of Eimeria, it is very necessary to plan the alternative control strategies (Chapman, 1997). Live vaccines containing virulent or attenuated strains of Eimeria are available. However, their use is limited in poultry industry owing to high cost (Subramanian et al., 2008). Additionally, these vaccines consist of several live Eimeria species, which make them labors as well as cost intensive to produce (Vermeulen, 1998). Hence, new strategies such as vaccination with affinity-purified antigens (CoxAbic®, Novartis, USA), recombinant proteins and chimeric improvement of poultry immunity are now actively pursued (Wallach et al., 1992; Wongi et al., 2002). Many studies demonstrated various levels of protection by vaccination with recombinant antigen or DNA vaccines against coccidiosis (Ding et al., 2005; Subramanian et al., 2008; Wu et al., 2004; Xu et al., 2006). It has also been found that DNA vaccines or recombinant antigen can provoke both humoral and cellmediated immune responses (Kim et al., 1989; Oshop et al., 2002; Song et al., 2000; Subramanian et al., 2008; Yang et al., 2010) and co-delivery of cytokines as adjuvants could enhance the potential for DNA vaccines or recombinant antigen to induce broad and longlasting humoral and cellular immunity (Ding et al., 2004; Song et al., 2010). Micronemes are the smallest of the apical organelles, structurally and functionally conserved in all apicomplexans (Bumstead and Tomley, 2000). Proteins secreted by microneme promote the attachment of parasites to the potential host cells (Tomley et al., 1996; Tomley and Soldati, 2001) and thus play a crucial role in the invasion process of apicomplexan parasites. Many proteins are secreted from the apical tip of the sporozoites, which help the Eimeria sporozoites to attach with gut epithelial cells and help in
et al., 2005). Some internalization of the sporozoites (Labbe microneme organelles have been described in some Eimeria species such as EtMIC2 was cloned by immunoscreening of a cDNA expression library (Tomley et al., 1996), EtMIC3 was cloned by screening a sporozoite cDNA library with two independent
et al., monoclonal antibodies raised against the oocyst stage (Labbe 2005). Eimeria brunetti is a species of Eimeria that causes hemorrhagic intestinal coccidiosis with bloody diarrhea, poor feed conversion, weight loss, and mortality in severe cases in young poultry. Lesions are limited to lower part of small intestine. So far, very few genes of this coccidian have been identified and evaluated for their immunogenicity. This present research of novel antigen and evaluated their protective efficiency against challenge with E. brunetti will open the development of a new approach of vaccines against this parasite. In this study, we described molecular cloning, expression and characterization of microneme-2 gene of E. brunetti isolated from an outbreak sample in Jiangsu, China. We also analyzed the evaluation of the humoral immune responses of chickens after vaccination with recombinant EbMIC2. 2. Materials and methods 2.1. Designing primers The PCR primers for applications of E. brunetti Microneme-2

(EbMIC-2) were designed by Primer Premier 5.0 software according to mRNA sequence published in GenBank (accession number AB723700). The sequence of the forward primer and reverse primers are as follows: 50 -GAATTCATGGCGCGCACTTTTTCT-30 and 50 -AAGCTTTTATGCTTCGGACGACGC-3’. Underline represents the EcoRI and HindIII enzymes site in the forward and reverse primers, respectively. 2.2. Animals and parasites New-hatched, free coccidian-yellow chickens were established from Jiangsu, PR. China and raised in clean brooder cages separately. Chicken Eimeria infection status were observed periodically by microscopic examination of feces and polymerase chain reaction (PCR) assay (Haug et al., 2007). Sporulated oocysts of E. brunetti JS strain were isolated from Jiangsu Province of China and maintained in Laboratory of Veterinary Molecular and Immunological Parasitology, stored in 2.5% potassium dichromate solution (K2Cr2O7) at 4  C and passages through chickens every 3 months interval. For infections, sporulated oocysts were washed and then purified according to the method described by (Zhao et al., 2001) followed by extensive washing several times with de-ionized water, the oocysts concentrations adjusted to the desired levels and used immediately. 2.3. Polyclonal immune sera against E. brunetti To produce polyclonal antibody, about 0.3 mg of the purified recombinant EbMIC2 protein was mixed with Freund's complete adjuvant (1:1 mixture) for the first injection into SD rats (Qualitied Certificate: SCXK 2008-0004; Experimental Animal Center of Jiangsu, PR China) subcutaneously in multiple sides. The same dose of purified recombinant EbMIC2 protein mixed with Freund's incomplete adjuvant was given after two weeks. Then a booster was given in the same conditions and then, the rats were reboosted again at intervals of 1 week. Finally, the serum was collected and stored at 20  C until used. Sera collected before protein injection was used as negative sera (Yanming et al., 2007). The polyclonal sera against chicken (chicken antisera) were collected from chickens experimentally infected with E. brunetti one week post infection. 2.4. Total RNA extraction Total RNA was isolated from 1.0  107 sporulated oocysts of E. brunetti as described (Carson et al., 2012; Sambrook et al., 2002). All the materials and equipments were treated with diethyl pyrocarbonate (DEPC) water overnight. The oocysts were grounded using a pre-chilled mortor and pestle, Trizol (Invitrogen) was added and homogenized for 30 min at room temperature. 200 ml Trichloromethane was added, and the mixture was spun at 12,000 rpm for 15 min at 4  C. Then RNA was precipitated from the supernatant by addition of isopropyl alcohol. The RNA was pelleted and dried then resuspended and washed by 70% ethanol. The pellet was suspended in DEPC water. The concentration was estimated by Nano-drop analysis and used in subsequent cDNA preparations immediately. 2.5. Reverse transcription and amplification of cDNA (RT-PCR) The cDNA was synthesized by reverse transcription reaction using cDNA Kit (SigmaeAldrich, USA) according to the manufacturer's instructions. Then cDNA products were stored at e 20  C until required. For the amplification of EbMIC2, 50 volume of PCR reaction was

T.D. Hoan et al. / Experimental Parasitology 162 (2016) 7e17

performed in a 0.5 ml micro-tube as below: 2 ml of cDNA template; 8 ml of dNTP Mixture 2.5 mM; 2 ml of Forward primer; 2 ml of Reverse primer; 5 ml of 10  LA PCR Buffer II; 0.5 ml of TaKaRa LATaq; De-ionized water up to 50 ml. The PCR reaction of Thermocycling condition as follows: 1 cycle at 94  C (5 min) for denaturing, followed by 30 cycles at 94  C (30 s), 58  C (30 s), 72  C (1 min) and a last cycle for elongation at 72  C for 10 min. Amplification was performed using the automated cycler (Bio-Rad, CA, USA). PCR € ttingen, products were separated by electrophoresis (Bio-metra, Go Germany) by loading onto 1.0% agarose gel (Peqlab, Erlangen, Germany). The gels were stained in an aqueous ethidium bromide (Golden view) solution (0.5 mg/ml) and DNA bands were visualized under UV light (transillumi-nator; UV wavelength, 254 nm; TFX20 M, Vilber Lourmat, France) and photographed by a digital camera (CSE-0028, Cybertech, Berlin, Germany) (Jenkins et al., 2006; Tsuji et al., 1997).

9

sonication (Vibra Cell; UK). The recombinant His6 tagged EbMIC2 was purified from the soluble fraction of the lysate using Hi-Trap metal chelating column (GE Healthcare, USA). The purified protein was dialysed extensively against PBS to remove imidazole. Yield of the affinity-purified protein was estimated using Bicinchoninic Acid kit (SigmaeAldrich, USA). Purified recombinant EbMIC2 protein was visualized on 12% SDS PAGE after staining with Coomassie brilliant blue stain and was stored in aliquots at 20  C until further analysis. Native EbMIC2 was purified from whole-cell lysates by method as described by Lock et al. (1988), using serial chromatography on DEAE-cellulose, Sephacryl S-200, Amicon Red-A gel and hydroxylapatite and then determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. 2.9. Immuno-blot for the recombinant EbMIC2 and native protein of EbMIC2

2.6. Cloning of EbMIC2 gene The ORF of E. brunetti MIC2 gene was amplified by PCR amplification and product was recovered using “AxyPrep™ DNA Gel Extraction kit” (Axygen, USA) according to manufacturer's instructions. Then purified fragment was ligated to pMD19-T cloning vector (Takara Co., Ltd., Dalian, China) following the manufacturer's recommendations and the ligation mixture was used to transform into Escherichia coli DH5a strain (Wang et al., 1995). The positive colonies were picked from plates containing 100 mg/ml ampicillin then cultured into 3e5 ml LB containing 100 mg/ml ampicillin. The positive clones were confirmed by digestion with EcoRI and HindIII enzymes and electrophoresis, selected recombinant clones were sequenced by Invitrogen Bio-tech (Shanghai Huajin Gene Bio-tech Co. Ltd., PR China) and sequence data was assembled and analyzed by DNAssist software version 2.2. The correction of EbMIC2 gene was then cloned into EcoRI/HindIII sites of pET28a (þ) vector (Novagen, USA). The recombinant plasmid was sequenced to confirm the EbMIC2 was inserted in the proper reading frame. 2.7. Sequence analysis Sequence similarity was analyzed using the BLASTP and BLASTX (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi). Microneme protein sequences were aligned using CLUSTALW1.8. The signal peptide, secondary structure and protein motifs were predicted using approaches accessible on the Internet: SignalP (http://www.cbs.dtu. dk/services/SignalP/), PSIpred (http://www.bioinf4.cs.ucl.ac.uk: 3000/psipred/), Motifscan (http://www.myhits.isb-sib.ch/cgibin/ motif_scan), respectively. One possible phylogenetic tree was constructed by aligning the amino acid sequences of the specific motif T and motifs 1, 2 and A through E using the neighbor-joining method (Saitou and Nei, 1987). The analyses were done using MEGA version 5.1 (http:// www.megasoftware.net) (Kumar et al., 2004). 2.8. Expression; purification of recombinant and native EbMIC2 Recombinant pET28a-MIC2 plasmid was transferred into competent E. coli BL21 strain and the protein expression in E. coli was induced using 0.8 mM of isopropyl-b-D-thiogalactopyranoside (IPTG; SigmaeAldrich, USA) at OD600 ¼ 0.6 at 37  C for culturing the cell. The induced bacterial cells were incubated at 37  C for 6 h after adding IPTG and were reaped by centrifugation, then cell lysates were prepared by sonication and analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For purification of recombinant protein, cell pellet was lysed using lysozyme (10 mg/ml) (SigmaeAldrich, USA) followed by

Samples including crude somatic extracts of E. brunetti sporozoites and the recombinant EbMIC2 protein was separated by 12% SDS-PAGE and transferred onto nitrocellulose transfer-membrane (Millipore, USA). After being blocked with 5% skimmed milk powder in TBS-Tween 20 (TBST). The membranes were incubated with the primary antibodies (rat antisera and chicken antisera, respectively) for 1 h at 37  C (dilution 1:100). Then Horseradish peroxidase (HRP)-conjugated goat anti-rat IgG, and HRP-conjugated donkey anti-chick IgG (Sigma, USA) were added, respectively, and incubated for 1 h at 37  C (1:5000 dilution). Finally, the bound antibody was detected using 3,3'-diaminobenzidine tetra hydrochloride (DAB) kit (Boster Bio-technology) according to manufacturer's instructions. 2.10. Construction of eukaryotic expression plasmid The 876 bp of EbMIC2 was inserted into the eukaryotic expression vector to evaluate its immunogenicity. The pMD-19T plasmid containing EbMIC2 and the pVAX1 vector (Invitrogen, Life Technologies) were digested with EcoRI and HindIII enzymes, then the EbMIC2 was ligated with the pVAX1 vector. The recombinant plasmid pVAX1-EbMIC2 was digested with the same restriction enzymes above and sequenced by Invitrogen bio-tech (Shanghai Huajin Bio-tech Co LTD, PR China). The recombinant plasmid pVAX1-EbMIC2 using as DNA vaccines were prepared using Qiagen Plasmid DNA Mid Kit (Qiagen, USA) according to the manufacturer's instructions. The eluted products were dissolved in TE buffer and diluted up to a concentration of 1 mg/ml, and stored at 20  C until required. 2.11. Detection of the expressions of proteins by eukaryotic expression plasmid in vivo The dose rate of 100 mg recombinant plasmid pVAX1-EbMIC2 was injected by intramuscularly for experimental chickens. One week post immunization later, injected tissues were collected and total RNA was extracted. The contaminating genomic DNA or plasmids injected were removed by treating with RNase-free DNase I (TaKaRa, China). RT-PCR assays were performed with cloning primer pairs of EbMIC2 gene as above (Heading 2.1). The PCR products were identified by electrophoresis onto 1% agarose gel. Western blot detection was performed as described by (Xu et al., 2008). Briefly, seven days after challenge, injected muscles were grinded and treated with ice-cold RIPA solution (0.1 M phenylmethyl-sulfonyl fluoride, 150 mM sodium chloride, 1% Nonnidet P-40, 0.1% SDS, 50 mM TriseHCl). Meanwhile the same site muscles from non-injected and pVAX1 plasmid injected chickens were

10

T.D. Hoan et al. / Experimental Parasitology 162 (2016) 7e17

collected for control. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane (Millipore, USA). The membrane was incubated with rat anti-EbMIC2 polyclonal antibody as primary antibody and horseradish peroxidase (HRP)-conjugated goat anti-rat IgG (Sigma) as secondary antibody. The bound antibody was detected using 3,3'-diaminobenzidine (DAB) according to the manufacturer's instructions.

chickens from unchallenged control group, pVAX1 control group and immunized groups were bled by cardiac puncture and the serum was used for cytokines and antibody analysis. The other 20 chickens in each group except the unchallenged control group were inoculated orally with 1  105 sporulated oocysts of Jiangsu strain while the unchallenged control group were given PBS orally. Seven days after challenge, all chickens were weighed and slaughtered for ileum collection.

2.12. Determination of serum antibody level of EbMIC2

2.15. Evaluation of immune protection

The IgG antibody levels against E. brunetti microneme 2 antigen in the serum were determined by ELISA as described (Lillehoj et al., 2005a). Briefly, flat-bottomed 96 wells of plate (Marxi-Sorp, Nunc, Denmark) were coated overnight at 4  C with 100 ml per well of the solution of soluble antigen of E. brunetti (10 mg/ml) in 0.05M carbonate buffer, pH 9.6. The plates were washed with 0.01M PBST and blocked with 5% bovine serum albumine (BSA) in PBST for 2 h at 37  C, followed by incubating for 2 h at 37  C with 100 ml of the serum samples with 1:100 of dilution in PBST with 5% BSA in duplicate. After washing three times, the plates were incubated for 2 h at room temperature with 100 ml/well of horseradish peroxidase-conjugated anti-chicken IgG antibody (Sigma) with 1:5000 of dilution with PBST in 5% BSA. The ELISA was developed by chromagen 3,30 ,5,5’-tetramethylbenzidine (TMB, Sigma, USA) and the optical density at 450 nm (OD450) was determine by a microplate spectophtometer.

The efficacy of immunization was evaluated on the basis of lesion score, body weight gain, oocyst output, oocyst decrease ratio and anti-coccidial index (ACI). Body weight gain of chickens in each group was determined by the body weight of the chickens at the end of the experiments subtracting the body weight at the time of challenge. Lesion scores were evaluated as described previously (Johnson and Reid, 1970). Additionally, the ileum content for each group was collected separately and oocysts per gram of content (OPG) were determined using McMaster's counting technique (Subramanian et al., 2008). Oocyst decrease ratio was calculated as follows: the number of oocysts from the challenged control chickens  vaccinated chickens/the challenged control chickens  100%. ACI was calculated as follows: (relative rate of weight gain þ survival rate)  (lesion value þ oocyst value).

2.13. Determination of serum cytokine concentration

Body weight gain, lesion score, fecal oocyst output, oocyst decrease ratio and anti-coccidial index (ACI) were expressed as means ± SD and were performed using the SPSS statistical package (SPSS for Window 20.0, SPSS Inc., Chicago, IL, USA). Differences among groups were tested with the one-way ANOVA Duncan test, mean values (- Value of less than 0.05 was considered significant).

The concentration of interferon-g (IFN-g), tumor growth factorb (TGF-b) interleukin-4 (IL-4), interleukin-10 (IL-10) and interleukin-17 (IL-17) in serum were detected by utilizing an indirect ELISA with the “chick cytokine ELISA Quantitation Kits” of CUSABIO life science, USA in duplicate according to manufacturer's instructions.

2.16. Statistical analysis

3. Results

2.14. Immunization and challenge experiment

3.1. Cloning of EbMIC2 gene

Fourteenth-day-old chickens were weighed and randomly separated into five groups of 30 each as shown in Table 1. Chickens in the unchallenged control group and challenged control group were injected with TE buffer (10 mM TriseHCl pH 8.0 and 1 mM EDTA) at the same injection site. Experimental groups were respectively injected by intramuscular leg with 100 ml plasmid pVAX1-EbMIC2 and recombinant EbMIC2 protein (concentration 1 mg/ml) at 14 days of age. The pVAX1 plasmid was also used as the vector control group; challenged and unchallenged control groups were injected with sterile TE at the same injection site. A booster immunization was given after 1 week of the first immunization with the same dose. After seven days post the second injection, 10

The products of E. brunetti MIC2 gene were successfully isolated by PCR amplification with specific primers and a fragment of the correct size of 876 bp was obtained (Fig. 1). The recovered PCR product was purified and successfully cloned into pMD19-T cloning vector which was confirmed by PCR identification and endonuclease digestion with EcoRI and HindIII enzymes (Fig. 2a and b). The sequence result of EbMIC-2 and corresponding amino acids compared with published sequence in GenBank (AB723700) were described in Fig. 3, displayed 99.43% similarity in the nucleotide sequence and 98.63% in amino acids sequence. It indicated that the cloned sequence was highly related to that one in GenBank.

Table 1 Effects of EbMIC2 against E. brunetti challenge on different parameters. Groups

Average body weight gain (g)

Unchallenged control Challenged control pVAX1 control pVAX1-EbMIC2 Recombinant EbMIC2 protein

132.43 78.28 89.71 125.03 106.72

± ± ± ± ±

9.97a 16.43c 15.59c 15.05a 15.32b

Mean lesion score (mean ± SD)

Oocyst output (x105) (mean ± SD)

Oocyst decrease ratio (%)

Anti-coccidial index (ACI)

0d 2.43 2.27 1.03 1.93

0e 5.07 3.86 1.14 2.53

100 0 23.89 77.43 50.05

200 131 142 179 159

± ± ± ±

0.62a 0.53a 0.85c 0.56b

± ± ± ±

2.07a 1.47b 0.50d 1.02c

Note: The values represent mean ± SD for 20 chickens. Oocyst decrease ratio was determined as percentage of non-immunized and unchallenged group. Values with in column not sharing a common superscript letter (aee) are significantly different at p > 0.05.

T.D. Hoan et al. / Experimental Parasitology 162 (2016) 7e17

The sequence results revealed that the ORF sequence of Jiangsu EbMIC2 strain was 876 bp. The open reading frame started with ATG at the beginning and ended with a TAA stop codon at base 876, encoding an about 32 kDa polypeptide of 291 amino acids. The deduced protein contains 38 basic amino acids residues (12 arginines and 26 lysines), and 31 acidic amino acids residues (14 glutamic and 17 aspartic acids). Meanwhile the total net charge was positive. There were 4 amino acids changes at 69 (Val/Leu), 281 (Ser/Pro), 284 (Ala/Val) and 286 (Arg/Gly). The theoretical pI of the protein was 4.60 and no signal peptide was found in the deduced protein, but one glycosylation site and twenty-one phosphorylation sites were detected by sequence analysis. Phylogenetic comparison of the deduced nucleotide sequence of EbMIC2 gene of Jiangsu strain with the nucleotide sequence of EbMIC2 on GenBank and other chicken coccidian (Eimeria tenella and Eimeria maxima) demonstrated that each MIC2 sequence clustered depending on their genus (Fig. 4). The correct fragment of EbMIC2 after sequencing was then inserted into EcoRI/HindIII sites of pET28a (þ) vector. The recombinant plasmid was confirmed with EcoRI and HindIII restricted enzymes digestion (Fig. 5a) and PCR identification (Fig. 5b). After digestion with EcoRI and HindIII, recombinant pET28a-EbMIC2 produced a fragment of approximately 876 bp which is equal to molecular mass of EbMIC2 gene and a PCR product of about 876 bp was obtained. The results showed that EbMIC2 was successfully ligated into pET28a vector. 3.2. Expression and purification of EbMIC2 protein The recombinant EbMIC2 expressed in E. coli BL21 cells as a double His 6 tagged fusion protein was purified. A protein band of 36 kDa size was detected in SDS-PAGE after staining with coomassie brilliant blue (Fig. 6a) and (Fig. 6b). Because of the 4 kDa fused protein in the pET28a (þ) vector, the recombinant protein's molecular weight was almost the value 32 kDa of rEbMIC2 calculated according to the deduced amino acid sequence. 3.3. Immunoblot for the recombinant EbMIC2 and native protein of EbMIC2 The results of the immunoblot assay indicated the recombinant protein was recognized by immusera of chickens infected with E. brunetti but no protein of anti-EbMIC2 in negative control was identified by normal chicken serum (Fig. 7). Rat anti-EbMIC2 antiserum bound to band of about 36 kDa in the somatic extract of E. brunetti sporozoites while no protein of anti-EbMIC2 in

Fig. 1. PCR amplification products of EbMIC2. Lane M e DL2000 DNA marker; Lane 1, 2, 3, 4 e EbMIC2 (876 bp) cDNA.

11

Fig. 2. a. Identification of recombinant pMD-EbMIC2 plasmid by restriction enzyme digestion. Lane M e DL2000 DNA marker; Lane 1 e Product digested by EcoRI and HindIII b. Identification of recombinant pMD-EbMIC2 plasmid by PCR amplification. Lane M e DL2000 DNA marker, Lane 1, 2 e PCR products (876 bp).

negative control was detected by normal rat serum (Fig. 8). 3.4. Identification of recombinant plasmid and the expression of EbMIC2 gene in vivo A fragment of approximately 876 bp of recombinant plasmid pVAX1-EbMIC2 was identified by digestion with EcoRI and HindIII (Fig. 9). This revealed that recombinant plasmid pVAX1-EbMIC2 was successfully constructed. Samples of muscle from chickens were analyzed by the RT-PCR with specific primers as above and gel electrophoresis. Results of RT-PCR indicated that the target fragment of EbMIC2 was determined from muscle RNA samples of chickens injected with pVAX1EbMIC2 (Fig. 10). No amplification products were generated from the respective non-vaccine injected controls. Western blot detection of the muscle of chickens injected with pVAX1-EbMIC2 indicated a prominent band of 36 kDa, which revealed the expression of EbMIC2 gene (Fig. 11). In contrast, no corresponding band was detected in the muscle of chickens injected with pVAX1 plasmid only. 3.5. Protective effects of EbMIC2 against E. brunetti challenge The immunizing efficacies of the EbMIC2 were described in Table 1. In the present study, no chicken died in any coccidial challenge group. There were no significant differences among five groups of the body weights of chickens at the day of immunization and challenge. After challenge, non-immunized challenged control groups exhibited significantly reduced weight gain compared with all vaccinated groups and the unchallenged control group chickens (p < 0.05). The weight gain of chickens immunized with recombinant EbMIC2 was significantly higher than that of challenged control and pVAX1 control, but lower than that of the unchallenged control and pVAX1-EbMIC2 immunized group. Chickens immunized with recombinant EbMIC2 and pVAX1EbMIC2 presented significantly lower oocyst output and higher oocyst decrease ratio compared with the pVAX1 and challenged controls (p < 0.05). Significant alleviations in ileum lesions score were also observed in immunized chickens. The results of the present study revealed that ACI in chickens group immunized with pVAX1-EbMIC2 (179) was higher than that of recombinant EbMIC2 vaccinated group (159). In case of chickens injected with pVAX1

12

T.D. Hoan et al. / Experimental Parasitology 162 (2016) 7e17

Fig. 3. Nucleotide sequence of EbMIC2 and corresponding amino acids sequence for Jiangsu strain and published sequence in GenBank. Letters indicated substituted and analyzed bases.

and challenged control groups showed considerably lower ACI (142 and 131, respectively). 3.6. IgG and cytokine levels in sera of immunized chickens The results in Fig. 12 showed that, serum from chickens immunized with recombinant EbMIC2 protein and plasmid pVAX1EbMIC2 showed significant higher level of IgG antibody (p < 0.05) compared with control groups. IFN-g, IL-10 and IL-17 of chickens in the two immunized groups all showed significant higher levels. However, TGF-b and IL-4 levels in the immunized groups were not significantly higher than that of pVAX1 control group, but significantly higher than that of the unchallenged group. 4. Discussion Microneme proteins are the main adhesions involved in the

attachment to the host cell surface by apicomplexans (Brossier and David Sibley, 2005). E. brunetti is one of the important pathogens of chicken coccidiosis. EbMIC2 sequence was published in the GenBank (Accession number AB723700). However, there were only a few reports about its character and immunogenicity. In the current study, our strategy was to isolate EbMIC2 from sporozoites oocyst of E. brunetti successfully with a high degree of similarities at 99.43% (nucleotide sequence) and 98.63% (amino acid sequence), respectively, revealed by comparing the sequence of EbMIC2 for Jiangsu strain with published EbMIC2 sequence of GenBank. Analysis of EbMIC2 sequences from different geographical areas will help better understand the nature of this gene. Microneme-2 protein from E. brunetti has 77% similarity with EmMIC2 and 71% with EtMIC2 in their amino acids sequence, respectively. The homology of E. brunetti microneme- 2 protein compared with the known species of Eimeria revealed that it was highly identical to some other DNAs consisting of microneme protein - 2 present at the

T.D. Hoan et al. / Experimental Parasitology 162 (2016) 7e17

13

E.tenellaMIC2/7h AF111703 E.tenellaMIC2 AF111702 E.tenellaMIC2 AF111839 E.tenellaMIC2 FJ807654 E.maximaMIC2Ht FR718971 E.brunettiMIC2 AB723700 E.brunettiMIC2Js E.maximaMIC7Ht FR718975 E.tenellaMIC4 AJ306453 E.tenellaMIC1 EU093966 E.maximaMIC5Ht FR718974 E.tenellaMIC5 AJ245536 E.maximaMIC3Ht FR718972 E.tenellaMIC3Ht FJ374765 E.tenellaMIC3 AY512381

1.5

1.0

0.5

0.0

Fig. 4. Phylogenetic tree of amino acids sequences between EbMIC2 genes. The phylogenic tree was constructed using MEGA version 5.10 by the neighbor-joining method with 500 bootstrap replicates. The bar represents a genetic distance of 0.5.

Fig. 5. a. Identification of pET28a-EbMIC2 recombinant plasmid by restriction enzyme digestion. Lane M e DL2000 DNA marker; Lane 1 e Product digested by EcoRI and HindIII. b. Identification of pET28a-EbMIC2 recombinant plasmid by PCR amplification. Lane M e DL2000 DNA marker; Lane 1 e PCR product (876 bp).

database of GenBank. This high percentage identity and homology give an indication about its immunogenic level among the Eimeria species. This demonstrated that it may prove a vaccine target against mixed infection of Eimeria spp. The phylogenetic analysis of the deduced nucleotide sequence of the EbMIC2 gene of Jiangsu strain with the nucleotide sequence of EbMIC2 on GenBank and other apicomplexan parasites such as E. tenella (EtMIC2) and E. maxima (EmMIC2) also demonstrated that each MIC2 sequence clustered depending on their genus. The results of cloning and sequence analysis revealed that the ORF sequence of EbMIC2 Jiangsu strain was 876 bp. The open reading frame (ORF) started with ATG at the beginning and ended with a TAA stop codon at base 876, encoding about 32 kDa polypeptide of 291 amino acids. The nucleotide sequence of the E. brunetti Jiangsu strain compared with published EbMIC2 sequence from GenBank revealed five change positions at 205, 240,

Fig. 6. a. SDS-PAGE analysis of the expressed products of recombinant pET28-EbMIC2 plasmid. Lane M e Protein molecular marker; Lane 1 e Induced product of pET28aEbMIC2 respective at 37  C before adding IPTG; Lane 2 e Induced product of pET28a-EbMIC2 respective for 2 h at 37  C after adding IPTG; Lane 3 e Induced product of pET28a-EbMIC2 respective for 4 h at 37  C after adding IPTG; Lane 4 e Induced product of pET28a-EbMIC2 respective for 6 h at 37  C after adding IPTG. b. Purification of expressed recombinant pET28a-EbMIC2 plasmid. Lane M e Protein molecular marker; Lane 1 e Purified recombinant protein of pET28a-EbMIC2; Lane 2 e IPTG induced E. coli lysate of recombinant protein.

841, 851 and 856, and the amino acids showed four different positions at 69, 281, 284 and 286. It appears that the nucleotide and amino acids sequence of EbMIC2 are conserved between the strains of E. brunetti. The antigenic analysis of EbMIC2 was also performed through the DNAstar software version 2.0 for better understanding and prediction of its immunogenicity. The translated DNA sequence of E. brunetti MIC2 with standard code suggested that it was consisted of highly immunogenic amino acids. The antigenic index and MHC Motifs cell motif highlighted a very good combination, predicted to be act as good vaccine target. Since antigenic sites are typically located in areas of greatest local hydrophilicity, the HydropathyHopp-Woods method was also analyzed to study antigenicity of this antigen. In this study, the expressed product was exclusively associated with IPTG induced cells visualized under reducing conditions. The results of expressed product of recombinant EbMIC2 found that the size of recombinant EbMIC2 protein was approximately 36 kDa,

14

T.D. Hoan et al. / Experimental Parasitology 162 (2016) 7e17

Fig. 7. Immunoblot for the native protein of EbMIC2. Lane M e Protein molecular marker; Lane 1 e Recombinant protein EbMIC2 probed by serum of experimental chickens infected E. brunetti as primary antibody; Lane 2 e Recombinant protein EbMIC2 probed by serum of normal chickens as primary antibody.

Fig. 9. Endonuclease digestion of recombinant plasmid pVAX1-EbMIC2. Lane M e DL5000 DNA marker; Lane 1 e EcoRI and HindIII enzyme digested product of EbMIC2 and pVAX1 plasmid.

Fig. 10. Detection of expression of recombinant plasmid pVAX1-EbMIC2 in chicken muscle by RT-PCR. Lane M e DL2000 DNA marker; Lane 1 e pVAX1-EbMIC2 injected muscles showing transcription of EbMIC2; Lane 2 e pVAX1 plasmid injected muscle; Lane 3 e The negative control. Fig. 8. Immunoblot for the recombinant EbMIC2. Lane M e Protein molecular marker; Lane 1 e Somatic extraction of E. brunetti sporozoites probed by rat rEbMIC2 antisera as primary antibody; Lane 2 e Somatic extraction of E. brunetti sporozoites probed by serum of normal rat as primary antibody.

which was larger than the deduced size of 32 kDa, because of the 4 kDa fused protein in the pET-28a vector. Serum from chicks artificially infected with E. brunetti reacted with recombinant EbMIC2 protein ~36 kDa. This finding suggested that EbMIC2 protein was secreted during the course of parasite infection and could induce antibody responses of chicken. However, further blot analysis of Eimeria excretory products using antiserum raised against recombinant EbMIC2 protein together with immunolocalization studies are still required to detect exact sites of this protein expression in the coccidian parasite and the recombinant EbMIC2 protein could induce high humoral immune response in infected chickens of E. brunetti microneme-2 gene. The parameters of average body weight gain; mean lesion score; oocyst output; oocyst decrease ratio and anti-coccidial index (ACI), all observed a significant difference among the vaccinated groups compared with unvaccinated groups. This indicated that chickens immunized with pVAX1-EbMIC2 was a good

Fig. 11. Detection of expression of recombinant plasmid pVAX1-EbMIC2 in chicken muscle by western blotting analysis. Lane M e Protein molecular marker; Lane 1 e The muscle sample from chicken injected with pVAX1-EbMIC2; Lane 2 e The muscle sample from chicken injected with pVAX1 plasmid.

T.D. Hoan et al. / Experimental Parasitology 162 (2016) 7e17 b 3

Serum IgG

a

c

350.00

2.5

b

300.00

2

b

(pg/ml)

OD (450nm)

IFN-concentration

400.00

c

15

1.5 1

250.00 200.00 150.00 100.00

0.5

a

a

TE control

pVAX1 control

50.00

0

0.35

pVAX1-MIC2

b

b

12.00

(pg/ml)

0.20 0.15

ab

6.00 4.00

0.05

2.00 TE control

pVAX1 control

Recombinant pETMIC2

0.00

pVAX1-MIC2

TE control

IL-10 concentration

e

2500

b

pVAX1 control

Recombinant pET-MIC2

pVAX1-MIC2

IL-17 concentration

f

d

b

60.00

c

bc

8.00

0.10

0.00

pVAX1-MIC2

c

a

a

Recombinant pETMIC2

IL-4 concentration

d

10.00

0.25

(ng/ml)

a

pVAX1 control

TGF- concentration

c

0.30

70.00

a

TE control

0.00

Recombinant pET-MIC2

c

2000

(pg/ml)

(pg/ml)

50.00 40.00 30.00 20.00

a

1500 1000

a

a

b

500

10.00

0

0.00 TE control

pVAX1 control

Recombinant pET-MIC2

pVAX1-MIC2

TE control

pVAX1 control Recombinant pET-MIC2

pVAX1-MIC2

Fig. 12. Serum levels of EbMIC2 specific IgG and cytokine in chickens. Chickens were boosted with TE (negative control), pVAX1 plasmid (pVAX1 control), recombinant EbMIC2 protein and pVAX1-EbMIC2. The IgG titers are expressed as mean ± SD with respect to absorbance at 450 nm. The concentrations of IFNg, IL-4, IL-10 and IL-17 (mean ± SD) in rg/ml, TGF-b concentration (mean ± SD) in hg/ml. Bars with different lower-case letters are significantly different (p < 0.05). (a) IgG titers; The concentration of (b) IFNg; (c) TGF-b; (d) IL4; (e) IL-10 and (f) IL-17, respectively. Note: significant difference (p < 0.05) between numbers with different letters. No significant difference (p > 0.05) between numbers with the same letter.

protection and immunized with recombinant EbMIC2 was effective in imparting partial protection in a homologous challenge. No chicken died with homologous challenge infection. There may be two possibilities for this; one may be that the mortality is not the detriment of the E. brunetti; the other may be that challenge could be too weak to cause mortality. The body weight gain of challenged control group was reduced significantly as compared with the unchallenged control and the mean lesion score of the group of challenged control was 2.43, which indicated that the challenge dose was quite appropriate. The similar kind of observation was already made by Shah (2010) that no chicken was died following homologous coccidiosis challenge infection. Moreover, challenge infection caused severe intestinal lesion score, significant reduction of body weight and feed efficiency which were pinpointing towards the active disease faced by the positive control groups in this study. The homologous challenge was observed after immunization of recombinant EbMIC2 in experimental chickens groups, a significant difference increase in weight gain, post-challenge, among the immunized groups compared with non-immunized groups was

performed. The oocyst output upon challenge was considerably reduced in the vaccinated chickens. Though the reduction in oocyst output was nowhere near the complete protection level, the oocyst output upon challenge was considerably reduced in the vaccinated chickens more than 77%, reduction may help significantly reduce the oocyst burden in a chicken shed. Immunization with recombinant EbMIC2 was effective in imparting partial protection in a homologous challenge. The level of protection observed by us was similar to other subunit coccidiosis vaccines tested by various investigators (Jenkins, 1998; Lillehoj et al., 2005b). In other studies conducted in our laboratory, oocyst reduction was up to 75% in vaccine pcDNA-TA4-IL2 following the homologue challenge infection parallel to this study (Xu et al., 2008), in vaccine pVAX1-cSZ-2IL-2, oocyst reduction even up to 80% (Shah, 2010) and pVAX1-cSZJN1 was more than 77% (Zhu et al., 2012) and pVAX1-EbAMA1 was more than 74% (Hoan et al., 2014). Another study described challenge experiments in groups of chicks vaccinated with recombinant EtMIC3-MAR protein or EtMIC3-MAR DNA and found that EtMIC3 vaccination results are highly significant reductions in oocyst output after challenge infection and indicating that EtMIC3 should

16

T.D. Hoan et al. / Experimental Parasitology 162 (2016) 7e17

be considered as a good candidate antigen for future recombinant vaccine development (Lai et al., 2011). Our study were also in close agreement with findings at the assessment of another sub-unit vaccine in coccidiosis (Jenkins, 1998). The role of humoral immunity during coccidiosis was debatable with most evidence pointing to a minor function (Lillehoj and Trout, 1996; Yun et al., 2000). However, the number of recent studies have demonstrated that antibodies do play an important role in immunity against coccidiosis (Belli et al., 2004; Constantinoiu et al., 2008; Ding et al., 2004) and could combine immunization with 3-1E protein with immunity-related cytokine genes induced higher serum antibody response and better protective immunity against coccidiosis than 3-1E alone (Constantinoiu et al., 2008). IFN-g is marks of Th1 immune response and seems to be dominant in coccidiosis (Cornelissen et al., 2009; Lowenthal et al., 1997). IL-4, IL-10 and IL-17, the Th2-type cytokine, were known to regulate humoral immunity and to function more effectively as helper for B-cell activation (Hames and Glover, 1988; InagakiOhara et al., 2006). TGF-b, as a regulatory (iTreg) cytokine, plays important role in the immune response against Eimeria antigens (Song et al., 2010). The chickens immunized with recombinant EbMIC2 showed marked rises of concentrations of IFN-g, IL-10, IL17 and TGF-b. However, the level IL-4 concentration of animals immunized with this antigen was not increased. In the protective experiment, the chickens immunized with recombinant plasmid pVAX1-EbMIC2 also demonstrated significant difference of IgG antibody and cytokine levels compared to control groups. In case of the chicks immunized with the recombinant protein of EbMIC2 could produce antibody levels and TGF-b a little higher than that of chickens immunized with pVAX1 control and unchallenged control. On the contrary, no significant change of IL-4 concentration between immunized and unimmunized chicks, indicating that EbMIC2 could stimulate antibody response in the natural infection. Many publications demonstrated that co-delivery of cytokines as adjuvants could enhance the potential for DNA vaccines to induce strong humoral and cellular immunity (Lillehoj et al., 2005a; Song et al., 2010; Subramanian et al., 2008; Wongi et al., 2002). On the other side, a large number of surface and internal antigens from different species and developmental stages of Eimeria have been reported (Jang et al., 2010; Jenkins, 1998; Schaap et al., 2004). Identification of antigens specific to Eimeria life cycle stages conveying protective immunity is a pivotal step and very important in developing recombinant vaccine in the future (Geriletu et al., 2011; Ma et al., 2010; Song et al., 2013; Zhu et al., 2012). In this study, the EbMIC2 could stimulate high levels of humoral immunity in the protective experiment. These results suggested that it might be an effective candidate for DNA vaccines against E. brunetti. In summary, EbMIC2 was successfully cloned and expressed. Sequence analysis and BLASTN search revealed that the clone had high homology (99%) with the known gene of E. brunetti deposited in the GenBank database. EbMIC2 gene possesses better immunogenicity and could become a candidate E. brunetti antigen for use in the development of prophylactic vaccine against chicken coccidiosis. Results of animal challenge experiment showed that recombinant EbMIC2 protein could induce partial protection while the recombinant plasmid pVAX1-EbMIC2 showed good protection against homologous challenge in chicken. This is the first DNA vaccine described in E. brunetti, so that further study is necessary to understand more characterizations of this parasite. Further studies are necessary to understand more characterizations of this parasite as well as the key molecular mechanisms of invasion of this parasite into host cells to confirm molecular binding ligands and interactions.

Acknowledgments This work was supported by the National Natural Science Foundation of P.R China. Tran Duc Hoan was supported by Bacgiang Agriculture and Forestry University, Vietnam through the PhD overseas scholarship scheme. References Anders, P., Jørgen, W. Hansen, 2005. The Epidemiology, Diagnosis and Control of Poultry Parasites. The Royal Veterinary and Agricultural University and Food and Agriculture Organization of the United Nations, Copenhagen, Denmark and Rome, Italy. Belli, S.I., Mai, K., Skene, C.D., Gleeson, M.T., Witcombe, D.M., Katrib, M., Finger, A., Wallach, M.G., Smith, N.C., 2004. Characterisation of the antigenic and immunogenic properties of bacterially expressed, sexual stage antigens of the coccidian parasite, Eimeria maxima. Vaccine 22, 4316e4325. Brossier, F., David Sibley, L., 2005. Toxoplasma gondii: microneme protein MIC2. Int. J. Biochem. Cell Biol. 37, 2266e2272. Bumstead, J., Tomley, F., 2000. Induction of secretion and surface capping of microneme proteins in Eimeria tenella. Mol. Biochem. Parasitol. 110 (2), 311e321. Carson, S., Miller, B. Heather, Witherow, D. Scott, 2012. Molecular Biology Techniques a Classroom Laboratory Manual. Academic Press, London, Uk. Chapman, H., 1997. Biochemical genetic and applied aspects of drug resistance in Eimeria parasites of the fowl. Avian Pathol. 25, 221e224. Constantinoiu, C.C., Molloy, J.B., Jorgensen, W.K., Coleman, G.T., 2008. Antibody response against endogenous stages of an attenuated strain of Eimeria tenella. Vet. Parasitol. 154, 193e204. Cornelissen, J., Swinkels, W., Boersma, W., Rebel, J., 2009. Host response to simultaneous infections with Eimeria acervulina, maxima and tenella: a cumulation of single responses. Vet. Parasitol. 162, 58e66. Ding, X., Lillehoj, H.S., Dalloul, R.A., Min, W., Sato, T., Yasuda, A., Lillehoj, E.P., 2005. In ovo vaccination with the Eimeria tenella EtMIC2 gene induces protective immunity against coccidiosis. Vaccine 23, 3733e3740. Ding, X., Lillehoj, H.S., Quiroz, M.A., Bevensee, E., Lillehoj, E.P., 2004. Protective immunity against Eimeria acervulina following in ovo immunization with a recombinant subunit vaccine and cytokine genes. Infect. Immun. 72, 6939e6944. Donal, P. Conway, Elizabeth, M.M., 2007. Poultry Coccidiosis, Diagnostic and Testing Proceduces. Blackwell Publishing, Iowa, USA. Geriletu, Xu, L., Xurihua, Li, X., 2011. Vaccination of chickens with DNA vaccine expressing Eimeria tenella MZ5-7 against coccidiosis. Veterinary Parasitol. 177, 6e12. Hames, B.D., Glover, D.M., 1988. Molecular Immunology. Oxford Washington DC, London SW7 2AZ, UK. Haug, A., Thebo, P., Jens, G.M., 2007. A simplified protocol for molecular identification of Eimeria species in field samples. Vet. Parasitol. 146, 35e45. Hoan, T.D., Thao, D.T., Gadahi, J.A., Song, X., Xu, L., Yan, R., Li, X., 2014. Analysis of humoral immune response and cytokines in chickens vaccinated with Eimeria brunetti apical membrane antigen-1 (EbAMA1) DNA vaccine. Exp. Parasitol. 144, 65e72. Inagaki-Ohara, K., Dewi, F., Hisaeda, H., Smith, A., Jimi, F., Miyahira, M., 2006. Intestinal intraepithelial lymphocytes sustain the epithelial barrier function against Eimeria vermiformis infection. Infect. Immun. 74, 5292e5301. Jang, S.I., Lillehoj, H.S., Lee, S.H., Lee, K.W., Park, M.S., Cha, S., Lillehoj, E.P., Subramanian, B.M., Sriraman, R., Srinivasan, V.A., 2010. Eimeria maxima recombinant Gam82 gametocyte antigen vaccine protects against coccidiosis and augments humoral and cell-mediated immunity. Vaccine 28, 2980e2985. Jenkins, M.C., Miska, K., Klopp, S., 2006. Improved Polimerase chain reaction technique for determining the species Composition of Eimeria in poultry Litter. Avian Dis. 50, 632e635. Jenkins, M.C., 1998. Progress on developing a recombinant coccidiosis vaccine. Int. J. Parasitol. 28, 1111e1119. Johnson, J., Reid, W.M., 1970. Anticoccidial drug: lesion scoring techniques in battery and floor-pen experiment. Exp. Parasitol. 28 (1), 30e36. Kim, K.S., Jenkins, M.C., Lillehoj, H.S., 1989. Immunization of chickens with live Escherichia coli expressing Eimeria acervulina merozoite recombinant antigen induces partial protection against coccidiosis. Infect. Immun. 57 (8), 2434e2440. Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Briefings Bioinforma. 5, 150e163.
, M., de Venevelles, P., Girard-Misguich, F., Bourdieu, C., Guillaume, A., Pe
ry, P., Labbe 2005. Eimeria tenella microneme protein EtMIC3: identification, localisation and role in host cell infection. Mol. Biochem. Parasitol. 140, 43e53. Lai, L., Bumstead, J., Liu, Y., Garnett, J., Campanero-Rhodes, M.A., et al., 2011. The role of Sialyl Glycan Recognition in host tissue Tropism of the avian parasite Eimeria tenella. PLoS Pathog. 7 (10), e1002296. http://dx.doi.org/10.1371/ journal.ppat.1002296. Lillehoj, H., Ding, X., Quiroz, M., Bevensee, E., Lillehoj, E., 2005a. Resistance to intestinal coccidiosis following DNA immunization with the cloned 3-1E

T.D. Hoan et al. / Experimental Parasitology 162 (2016) 7e17 Eimeriagene plus IL-2, IL-15 and IFN-g. Avian Dis. 49, 112e117. Lillehoj, H.S., Ding, X., Dalloul, R.A., Sato, T., Yasuda, A., Lillehoj, E.P., 2005b. Embryo vaccination against Eimeria tenella and E. acervulina infections using recombinant proteins and cytokine adjuvant. J. Parasitol 91 (3), 666e673. Lillehoj, H.S., Trout, J.M., 1996. Avian gut-associated lymphoid tissues and intestinal immune responses to Eimeria parasites. Clin. Microbiol. Rev. 9, 349e360. Lock, R.A., Paton, J.C., Hansman, D., 1988. Purification and immunological characterization of neuraminidase produced by Streptococcus pneumoniae. Microb. Pathog. 4, 33e43. Lowenthal, J., York, J., O'Neil, T., Rhodes, S., Prowse, S., Strom, D., 1997. In vivo effects of chicken interferon-gamma during infection with Eimeria. J Interferon Cytokine Res 17, 551e558. Ma, D., Chunli, M., Long, P., Guangxing, L., Jinghong, Y., Jiehua, H., Haofan, C., Xiaofeng, R., 2010. Vaccination of chickens with DNA vaccine encoding Eimeria acervulina 3-1E and chicken IL-15 offers protection against homologous challenge. Exp. Parasitol. 127, 208e214. Morris, G., Gasser, R., 2006. Biotechnological advances in the diagnosis of avian coccidiosis and the analysis of genetic variation in Eimeria. Biotechnol. Adv. 24, 590e603. Oshop, G.L., Elankumaran, S., Heckert, R.A., 2002. DNA vaccination in the avian. Veterinary Immunol. Immunopathol. 89, 1e12. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406e425. Sambrook, J., Fritsch, Maniatis, T., 2002. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Schaap, D.C., Arts, G., Kroeze, J., Niessen, R., Roosmalen-Vos, S.V., Spreeuwenberg, K., Kuiper, C.M., Beek-Verhoeven, N.V.D., Kok, J.J., Knegtel, R.M.A., Vermeulen, A.N., 2004. An Eimeria vaccine candidate appears to be lactate dehydrogenase; characterization and comparative analysis. Parasitol 128, 603e616. Shah, M., 2010. Research on the Multi-epitopes DNA vaccines against chicken coccidiosis. Nanjing Agricultural, Nanjing. Song, H., Baofeng, Q., Ruofeng, Y., Lixin, X., Xiaokai, S., Xiangrui, L., 2013. The protective efficacy of chimeric SO7/IL-2 DNA vaccine against coccidiosis in chickens. Res. Veterinary Sci. 94, 562e567. Song, H., Xiaokai, S., Lixin, X., Ruofeng, Y., Muhammad, A., Shah, A., Xiangrui, L., 2010. Changes of cytokines and IgG antibody in chickens vaccinated with DNA vaccines encoding Eimeria acervulina lactate dehydrogenase. Vet. Parasitol. 173, 219e227. Song, K.D., Lillehoj, H.S., Choi, K.D., Yun, C.H., Parcells, M.S., Huynh, J.T., Han, J.Y., 2000. A DNA vaccine encoding a conserved Eimeria protein induces protective immunity against live Eimeria acervulina challenge. Vaccine 19, 243e252. Subramanian, M.B., Sriraman, R., Rao, H.N., Raghul, J., Thiagarajan, D., Srinivasan, V.A., 2008. Cloning, expression and evaluation of the efficacy of a recombinant Eimeria tenella sporozoite antigen in birds. Vaccine 26,

17

3489e3496. Tomley, F.M., Bumstead, J.M., Billington, K.J., Dunn, P.P.J., 1996. Molecular cloning and characterization of a novel acidic microneme protein (Etmic-2) from the apicomplexan protozoan parasite, Eimeria tenella. Mol. Biochem. Parasitol. 79, 195e206. Tomley, F.M., Soldati, D.S., 2001. Mix and match modules: structure and function of microneme proteins in apicomplexan parasites. Trends Parasitol. 17, 81e88. Tsuji, N., Kawazu, S., Ohta, M., Kamio, T., Isobe, T., Shimura, K., Fujisaki, K., 1997. Discrimination of eight chicken Eimeria species using the two-step polymerase chain reaction. Parasitol 83, 966e970. Vermeulen, A., 1998. Progress in recombinant vaccine development against coccidiosis - a review and prospects into the next millennium. Int. J. Parasitol. 28, 1121e1130. Wallach, M., Halabi, A., Pillema, G., Sor, S.O., Meucha, D., Lal, M., 1992. Maternal immunization with gametocyte antigens as a means of providing protective immunity against Eimeria maxima in chickens. Infect. Immun. 60, 2036e2039. Wang, X., Zhang, J., Li, F., 1995. Transformtion of Plasmid DNA into Bacteria Cells. Practical Protocols in Molecular Biology. Science Press, p. 57. Wongi, M., Lillehoj, H., Burnside, J., Weining, K., Staeheli, P., Zhu, J., 2002. Adjuvant effects of IL-1B, IL-2, IL-8, IL-15, IFN-a, IFN-g,TGF- B and lymphotactin on DNA. Vaccin. against Eimeria acervulina Vaccine 20, 267e274. Wu, S.-Q., Wang, M., Liu, Q., Zhu, Y.-J., Suo, X., Jiang, J.-S., 2004. Construction of DNA vaccines and their induced protective immunity against experimental Eimeria tenella infection. Parasitol. Res. 94, 332e336. Xu, Q., Song, X., Xu, L., Yan, R., Shah, M., Li, X., 2008. Vaccination of chickens with a chimeric DNA vaccine encoding Eimeria tenella TA4 and chicken IL-2 induces protective immunity against coccidiosis. Vet. Parasitol. 156, 319e323. Xu, S., Chen, T., Wang, M., 2006. Protective immunity enhanced by chimeric DNA prime-protein booster strategy against Eimeria tenella challenge. Avian Dis. 50 (4), 579e585. Yang, G., Wang, C., Hao, F., Zhao, D., Zhang, Y., Li, Y., 2010. Studies on construction of a recombinant Eimeria tenella SO7 gene expressing Escherichia coli and its protective efficacy against homologous infection. Parasitol. Int. 59, 517e523. Yanming, S., Ruofeng, Y., Muleke, C., Guangwei, Z., Lixin, X., Xiangrui, L., 2007. Vaccination of goats with recombinant galectin antigen induces partial protection against Haemonchus contortus infection. Parasitol. Immunol. 29, 319e326. Yun, C.H., Lillehoj, H.S., Lillehoj, E.P., 2000. Intestinal immune responses to coccidiosis. Dev. Comp. Immunol. 24, 303e324. Zhao, X., Duszynski, Donald W., Loker, Eric S., 2001. A simple method of DNA extraction for Eimeria species. Microbiol. Method 44, 131e137. Zhu, H., Yan, R., Wang, S., Song, X., Xu, L., Li, X., 2012. Identification and molecular characterization of a novel antigen of Eimeria acervulina. Mol. Biochem. Parasitol. 186, 21e28.