Molecular characterisation and the protective immunity evaluation of Eimeria maxima surface antigen gene

Molecular characterisation and the protective immunity evaluation of Eimeria maxima surface... Background: Coccidiosis is recognised as a major parasitic disease in chickens. Eimeria maxima is considered as a highly immunoprotective species within the Eimeria spp. family that infects chickens. In the present research, the surface antigen gene of E. maxima (EmSAG) was cloned, and the ability of EmSAG to stimulate protection against E. maxima was evaluated. Methods: Prokaryotic and eukaryotic plasmids expressing EmSAG were constructed. The EmSAG transcription and expression in vivo was performed based on the RT-PCR and immunoblot analysis. The expression of EmSAG in sporozoites and merozoites was detected through immunofluorescence analyses. The immune protection was assessed based on challenge experiments. Flow cytometry assays were used to determine the T cell subpopulations. The serum antibody and cytokine levels were evaluated by ELISA. Results: The open reading frame (ORF) of EmSAG gene contained 645 bp encoding 214 amino acid residues. The immunoblot and RT-PCR analyses indicated that the EmSAG gene were transcribed and expressed in vivo. The EmSAG proteins were expressed in sporozoite and merozoite stages of E. maxima by the immunofluorescence assay. Challenge experiments showed that both pVAX1-SAG and the recombinant EmSAG (rEmSAG) proteins were successful in alleviating jejunal lesions, decreasing loss of body weight and the oocyst ratio. Additionally, these + + experiments possessed anticoccidial indices (ACI) of more than 170. Higher percentages of CD4 and CD8 T cells were detected in both EmSAG-inoculated birds than those of the negative control groups (P < 0.05). The EmSAG- specific antibody concentrations of both the rEmSAG and pVAX1-EmSAG groups were much higher than those of the negative controls (P < 0.05). Higher concentrations of IL-4, IFN-γ, TGF-β1 and IL-17 were observed more in both the rEmSAG protein and pVAX1-SAG inoculated groups than those of negative controls (P < 0.05). Conclusions: Our findings suggest that EmSAG is capable of eliciting a moderate immune protection and could be used as an effective vaccine candidate against E. maxima. Keywords: Eimeria maxima, Surface antigen, Cytokines, Vaccine, Immunity * Correspondence: lixiangrui@njau.edu.cn College of Veterinary Medicine, Nanjing Agriculture University, 1 Weigang, Nanjing, Jiangsu 210095, People’s Republic of China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. Parasites & Vectors (2018) 11:325 Page 2 of 12 Background (Invitrogen Biotech, Shanghai, China) and verification, the Coccidiosis is recognised as a major parasitic disease in positive clones were confirmed as pET-32a/ EmSAG. chickens seriously affecting the efficiency of feed conver- sion and leading to decreased production. Eimeria Expression of the recombinant EmSAG protein maxima has been recognised as one of the most eco- The sequence identity of EmSAG was compared to the nomically significant species of Eimeria [1]. Currently, known SAG sequences of other Eimeria spp. and prophylactic chemotherapy with anticoccidial drugs is assessed using the BLASTx and BLASTp search tools the major control strategy for coccidiosis. Traditional (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The amino acid anticoccidial drugs and live vaccines have their own de- sequence of EmSAG was used to predict N-terminal sig- fect [2]. Subunit vaccines encoding the Eimeria proteins nal peptides through a bioinformatics online program which stimulated protective immunity were accepted as (http://www.cbs.dtu.dk/services/SignalP/). The clado- effective vaccines against coccidiosis [3–5]. Recently, gram was made using the MEGA 6.0 programme with many reports have shown that cell-mediated immunity the neighbour-joining method. The pET-32a/EmSAG could be stimulated by DNA vaccines [6–10]. was expressed in E. coli BL21 (DE3) as described previ- Surface antigens have been proven to confer protec- ously [21]. The recombinant protein was purified and tion against coccidiosis by altering key processes in host the concentration of the sample was determined using cell invasions [11]. The SAGs protein of Eimeria tenella the Bradford method [22]. The rEmSAG protein was is capable of inducing an immune response against coc- kept frozen (-70 °C) until further analysis. cidiosis in chickens [12]. Therefore, surface antigens and cell adhesion proteins have been suggested as promising Development of anti-EmSAG antibodies against the vaccine candidates against parasitic infections [13, 14]. rEmSAG protein Eimeria maxima is regarded as a highly immunopro- Rat polyclonal anti-EmSAG antibodies were generated tective species within the family of Eimeria spp. affecting in the Sprague-Dawley rats at 4 weeks of age. Rats were chickens [15–19]. In this study, subunit and DNA vac- subcutaneously immunised with a total of 0.3 mg of cines made from EmSAG were evaluated for their pro- rEmSAG protein mixed with Freund’s complete adju- tection against E. maxima. vant. Fourteen days after the first immunisation, the rats were given a booster injection with 0.3 mg of rEmSAG Methods protein in Freund’s incomplete adjuvant. Three booster Chickens and parasites doses were given at 1-week intervals. Finally, rat serum Eimeria-free birds at one day of age were reared in cap- containing antibodies were obtained after the last tivity with provided water and feed ad libitum. The birds booster injection and kept frozen (at -70 °C) until subse- were placed in a coccidia-free environment. The Jiangsu quent analysis. Pre-immunisation serum was obtained strain of E. maxima was developed and maintained in for later use as the negative control [23]. Eimeria-free birds our laboratory. Sporozoites from E. maxima oocysts were cleaned and sporulated as previ- Construction of eukaryotic plasmid of EmSAG ously described [20]. The construction of eukaryotic plasmid of EmSAG was conducted and purified as previously described [20]. Amplification and prokaryotic expression of EmSAG Briefly, the EmSAG fragment was inserted into the The construction of prokaryotic expression of EmSAG pVAX1, following the sequence analysis (Invitrogen Bio- was conducted as previously described [20]. Briefly, the tech) and verification, the positive clones were con- EmSAG-encoding sequence (GenBank: XM_013482011.1) firmed as pVAX1-EmSAG. The plasmids encoding was amplified by PCR. EmSAG-specific primers were uti- EmSAG were extracted using EndoFree Plasmid MEGA lised for the PCR assays: SAG1 (forward primer: 5'-CGC Kit (Qiagen, Valencia, CA, USA). The concentration of GGA TCC GAC ACA ATC TCC AGC CCT-3'; BamHI the sample was determined using as per the method sug- restriction sites underlined) and SAG2 (reverse primer: gested earlier [20]. Finally, the plasmids were kept frozen 5'-ATT GCG GCC GCT CAA ATG AGA ACA GAT (-20 °C) until subsequent analysis. GCG-3'; NotI restriction sites underlined) with E. maxima cDNA as a template. The amplification products of Immunoblot analysis of native EmSAG and rEmSAG proteins EmSAG cloned in pMD-19T (TaKaRa, Dalian, China) re- Immunoblot analyses for rEmSAG and native EmSAG sulted in the formation of recombinant plasmid were performed as described in an earlier work [20]. Rat pMD-19T-EmSAG. Subsequently, the EmSAG gene was anti-rEmSAG sera (dilutions of 1:200) were used to de- inserted into the pET-32a (+) (Novagen, Madison, WI, tect sporozoites. Chicken anti-E. maxima sera (dilutions USA) frame of expression vector system and confirmed by of 1:100) were used to detect the rEmSAG protein. Goat endonuclease digestion. Following sequence analysis anti-rat HRP-IgG and donkey anti-chick HRP-IgG Liu et al. Parasites & Vectors (2018) 11:325 Page 3 of 12 (Sigma-Aldrich, St. Louis, MO, USA) were used as a sec- Assessment of immune protection ondary antibody. The chickens were monitored for body weight gain and signs of immune protection (jejunal lesion score, survival rate and change in oocyst ratio). Lesion scrapings were Transcription and expression of pVAX1-EmSAG in vivo microscopically examined for any coccidia, whenever The EmSAG transcription in vivo was performed based there was doubt of truly coccidia-induced lesions. The je- on the RT-PCR and immunoblot analysis, as previously junal lesion scores of the birds were also evaluated, as de- described [24]. Briefly, in coccidia-free chickens, a total scribed in previous research [23]. The body weight gains of 100 μg pVAX1-EmSAG plasmid was intramuscularly were measured at different time-points: the days of vac- injected into the legs. In the pVAX1 control group, the cination, at the time of the coccidia challenge, and at the 100 μg pVAX1 plasmid was injected into the legs. One end of the test. All the jejunal contents from each bird week post-immunisation, the tissues from vaccinated were harvested and used to evaluate the oocyst counts as and non-vaccinated chickens were collected for both described in a previous study [26]. Using the McMaster’s RT-PCR and immunoblot analyses. EmSAG-specific counting method, oocysts and the oocyst ratio were primers were utilised for the RT-PCR assays. Rat assessed as previously described [27]. Anticoccidial index anti-rEmSAG sera (dilutions of 1:200) were used to de- (ACI) values were evaluated as per the standard formula tect pVAX1-EmSAG expression. The secondary antibody for assessing protection against E. maxima [20]. was HRP-conjugated goat anti-rat IgG (Sigma-Aldrich). ELISA analysis of the serum antibody and cytokine Location of the EmSAG protein in sporozoites and EmSAG-specific IgY/IgG antibodies were detected by merozoites stages ELISA using the rEmSAG protein as a coating antigen, Immunofluorescence technique was used to locate following previous protocols [28]. The serum samples EmSAG in sporozoites and merozoites as previously de- (1:50 dilution) were detected using the secondary anti- scribed [25]. Nuclei were probed with 2-(4-amidinophe- bodies of donkey anti-chicken HRP-conjugated IgG nyl)-6-indole carbamidinedihydrochloride (DAPI, monoclonal antibody (Sigma-Aldrich). The experiment Sigma-Aldrich). Rat anti-EmSAG sera (1:100 dilutions) was completed in duplicate. were used as the primary antibody. The secondary anti- For cytokines level analysis, serum samples were ob- body was Cy3-conjugated goat anti-rat IgG (Beyotime, tained and measured as previously described [29]. Briefly, Haimen, Jiangsu, China) (dilution of 1:1000). The slides 10 days after the last inoculation, the serum samples of the were analysed using fluorescence microscopy (Nikon, birds (n = 5) per group were harvested to evaluate the cy- Tokyo, Japan). tokines. The titers of IL-4, IL-17, IFN-γ and TGF-β1were measured using ELISA kits (CUSABIO, Wuhan, China). Experimental design The data was pooled from three independent experiments. Chickens at 14 days of age, negative for Eimeria were placed in six groups, each including 30 birds. The chick- Determination of T-cell response ens were inoculated intramuscularly injection with the The counts of T cells in the treatment groups were eval- pVAX1-SAG (100 μg/chick) or rEmSAG protein (200 uated by flow cytometry analysis as previously described μg/chick). In the pVAX1 control chickens, a total of 100 [30, 31]. Spleens were extracted from 5 chickens of each μg pVAX1 plasmid was injected into the legs. In the group at pre-, first-, and second-vaccination days. Lym- pET-32a control chickens group, a total of 200 μg phocytes were obtained from the spleens were stained pET-32a protein was injected as above. The challenged with SPRD-conjugated CD3 monoclonal antibodies. The and unchallenged control birds were immunised with cells were then probed with PE-conjugated mouse PBS. One week later, the birds were boosted with the monoclonal anti-chicken CD4 or the PE-conjugated same route as the primary immunisation. Subsequently, mouse monoclonal anti-chicken CD8 (Southern Biotech- 7 days after the last immunisation, 1 × 10 sporulated nology Associates, Birmingham, AL, USA). Using FACS oocysts of E. maxima were given to all the birds except flow cytometer, the stained cells were analysed with Cell the negative control birds. Seven days later, the birds Quest software (BD Biosciences, San Jose, CA, USA). were euthanised to measure their immune response and degree of coccidial protection. Moreover, the birds Statistical analysis (n = 5 per group) were placed in another coccidia-free All data was expressed as the mean ± standard deviation room. Finally, 10 days after the last immunisation, the using the SPSS Statistical Software (SPSS Inc., Chicago, serum samples were harvested and kept frozen (-20 °C) IL, USA). The data were analysed with one-way ANOVA until further antibodies and cytokine production ana- using Duncan’s post-hoc test and considered to be statis- lysis could be conducted. tically significant at P < 0.05. Liu et al. Parasites & Vectors (2018) 11:325 Page 4 of 12 Results EmSAG sequence analysis Using E. maxima cDNA as a template, the PCR product of EmSAG was isolated and ligated with pMD19-T. Se- quence analysis showed that the EmSAG ORF encoded a protein of 24.73 kDa with a pI of 4.808. As shown in Fig. 1, the phylogenetic tree formulation indicated that the kinship of EmSAG protein was highly related to the EtTA4 and EnNA4 when compared with other Eimeria spp. (E. mitis, E. brunetti, E. praecox, E. acervulina and E. necatrix). The amino acid sequence was analysed with the SignalP programme. The findings suggested an obvi- ous signal peptide possessed a cleavage site between pos- ition 21 and 22. Purification of the rEmSAG protein The pET32a/EmSAG plasmids were expressed in E. coli BL21. After IPTG induction, the rEmSAG proteins were harvested. The purified fusion rEmSAG protein was ap- proximately 43 kDa (Fig. 2). This calculated total value of 43 kDa was considered accurate as the sum of both the approximate 20 kDa length of pET-32a (+) and the approximate 23 kDa length of the EmSAG protein. Immunoblot analysis of native and rEmSAG proteins The native and rEmSAG proteins were evaluated by the western blot method (Fig. 3). The rEmSAG protein was tested using chicken E. maxima-specific antibodies, but not by the antibodies of unimmunised chickens. Further- more, the western blot assay also showed a band of al- most 26 kDa belonging to the sporozoites protein detected by rat anti-rEmSAG antibodies (Fig. 3), in con- trast to the serum from the negative control rats that did not display any bands. Fig. 2 Purified rEmSAG protein separated by SDS-PAGE. Lane M: pre-stained protein marker; Lane 1: the purified rEmSAG protein Identification of EmSAG location in sporozoites and stained with Coomassie brilliant blue merozoites The location of EmSAG in sporozoites and merozoites of E. maxima was confirmed using immunofluorescence analyses (Fig. 4). The EmSAG protein was detected using rat anti-rEmSAG antibodies, and Cy3-conjugated goat anti-rat IgG as secondary antibodies shown in red Fig. 1 The phylogenetic tree was constructed using CLUSTAL W alignment and neighbour-joining method of the software MEGA 6.0 Liu et al. Parasites & Vectors (2018) 11:325 Page 5 of 12 Fig. 3 Immunoblot analysis for native and rEmSAG proteins. Lane M: pre-stained protein marker; Lane 1: rEmSAG recognised by chick anti-E. maxima serum; Lane 2: rEmSAG protein tested against unimmunised chicken sera; Lane 3: E. maxima protein from sporozoites detected by rat anti-rEmSAG sera; Lane 4: protein of E. maxima sporozoites detected by unimmunised rat sera fluorescence, whereas no red fluorescence was detected in Analysis of T cell subpopulations cells probed with the pre-immune rat serum. The nuclei To evaluate the EmSAG specific T-cell responses, flow of the sporozoites and merozoites were visualised as blue. cytometry assays were used to analyse the CD4 and These results suggest that EmSAG was expressed in both CD8 T cells. The spleen lymphocytes were collected at the sporozoite and merozoite stages of E. maxima. pre-, first-, and second-inoculation time-points (Fig. 8). After the last vaccination, the percentage of CD4 in the EmSAG-immunised chickens was higher (ANOVA, Identification of transcription and expression of F = 46.28, P < 0.0001), than those in the PBS group, (4, 20) pVAX1-EmSAG in vivo pVAX1.0 group, and the pET-32a (+) group. Regarding Transcription and expression of pVAX1-SAG in vivo was CD8 T cells, EmSAG groups showed a higher evaluated through RT-PCR, using the EmSAG-specific (ANOVA, F = 43.59, P < 0.0001) percentage, (4, 20) primers. A specific DNA band was detected belonging to whereas the PBS, pVAX1 and pET-32a (+) control pVAX1-EmSAG in the tissues of the injected site (Fig. 5a, group remained at low levels after the second immun- Lane 4). The RNA samples from non-inoculated and isation (Table 1 and Fig. 8). pVAX1-inoculated tissues did not detect any band in the RT-PCR analyses (Fig. 5a, Lanes 1, 2 and 3). In addition, expression of pVAX1-SAG in vivo was de- Immune protection of EmSAG against E. maxima tected through immunoblot analysis. A unique band of To analyse the immune protection of EmSAG against E. approximately 26 kDa was detected in the maxima, the challenge experiments were assessed. The de- pVAX1-EmSAG-vaccinated muscle sample. In contrast, grees of immune protection conferred by vaccinations of no band was shown in the pVAX1-immunised muscle pVAX1-EmSAG and rEmSAG proteins were measured, samples (Fig. 5b). These results indicate the successful and the results of ACI are described in Table 2.Birds inoc- transcription and expression of the EmSAG gene in vivo. ulated with EmSAG exhibited higher weight gains (ANOVA, F = 27.67, P < 0.0001) and greater de- (5, 174) creases in oocyst ratios when compared to all other groups. Determination of IgY/IgG and cytokines levels using ELISA The ACIs of the EmSAG-immunised chickens were more To evaluate the titers of IgY/IgG and the cytokines, than 170, providing moderate protective immunity. serum samples from the immunised birds (n = 5 per group) were harvested at 10 days after the last vaccin- ation. The anti-EmSAG IgY/IgG titers of each group are Discussion shown in Fig. 6. The IgY/IgG titers of both In this research, both DNA and recombinant protein EmSAG-immunised groups were much higher (ANOVA, vaccines encoding EmSAG of E. maxima were compared F = 77.78, P < 0.0001) compared to the controls. regarding their abilities to induce protection against E. (4, 20) The titers of cytokines were measured using ELISA maxima infection. These results indicated that inocula- (Fig. 7). The serum samples in both pVAX1-EmSAG tion with EmSAG could promote IgG levels in the sera and rEmSAG-immunised chickens displayed higher ti- and upregulated the titers of IL-4, IFN-γ, IL-17 and ters of IFN-γ (ANOVA, F = 43.59, P < 0.0001), TGF-β1. Furthermore, the data from the animal experi- (4, 20) IL-17 (ANOVA, F = 42.25, P < 0.0001), IL-4 ments proved that EmSAG-immunised groups could (4, 20) (ANOVA, F = 3.25, P = 0.033) and TGF-β1 produce ACIs of more than 170. Taken together, these (4, 20) (ANOVA, F = 48.12, P < 0.0001) compared to the data demonstrate that EmSAG vaccines could stimulate (4, 20) negative controls. moderate protection against E. maxima. Liu et al. Parasites & Vectors (2018) 11:325 Page 6 of 12 Fig. 4 Expression of EmSAG protein in sporozoites and merozoites at 100× magnification. a The sporozoites were detected by rat anti-rEmSAG antibodies. a1 Sporozoites were dyed by Cy3. a2 The nuclei were probed by DAPI. a3 Overlaps of Cy3 and DAPI. b The sporozoites were detected by unimmunised rat antibodies. b1 Cy3 stains. b2 DAPI stains. b3 Merge. c Merozoites were detected by rat anti-rEmSAG antibodies. c1 Cy3 stains. c2 DAPI stains. c3 Merge. d The merozoites were detected by unimmunised rat antibodies. d1 Cy3 stains. d2 DAPI stains. d3 Merge. Scale-bars:10 μm DNA and recombinant protein vaccines were reported challenged with E. maxima when compared with to induce immuno-protection to live parasite challenge. non-vaccinated and parasite-challenged groups. Xu et al. Higher body weight gain, lower fecal oocyst shedding [33] determined that pcDNA3.0-TA4-IL-2 could de- and reduced intestinal pathology were detected for im- crease caecal lesions and body weight loss as well as pro- mune protection. Jang et al. [32] reported that birds had duce an ACI of 192. Song et al. [34] reported that lower oocyst concentration in droppings and reduced in- chickens immunised with pMP13 plasmid showed sig- testinal pathology after vaccination with Gam82 and nificantly lower number of oocysts following the Liu et al. Parasites & Vectors (2018) 11:325 Page 7 of 12 Fig. 5 Expression and transcription of pVAX1-SAG in vivo were identified through RT-PCR and western blot assays. a RT-PCR of pVAX1-EmSAG transcription in chicken muscle. Lane M: DNA marker DL2000; Lanes 1 and 2: the muscle RNA sample from the non-inoculated chicken; Lane 3: the muscle RNA sample from the pVAX1-inoculated chicken; Lane 4: the muscle RNA sample from the pVAX1-EmSAG injected chicken. b Western blot of pVAX1-EmSAG in chicken muscle. Lane M: pre-stained protein marker; Lane 1: the protein sample from pVAX1-inoculated chickens; Lane 2: the protein sample from pVAX1- EmSAG inoculated chickens challenge with E. acervulina compared to those in the protein exhibited higher specific antibodies concentra- negative controls. Similar results were detected in this tion than the negative controls. In this research, the anti- study, both pVAX1-EmSAG and rEmSAG vaccines were body titers of the EmSAG-immunised animals were successful in alleviating jejunal lesions, decreasing loss higher than the negative controls. The findings of this of body weight and the oocyst ratio. investigation confirmed that EmSAG could induce The chick-anti-Eimeria specific antibodies have been humoral immune response. previously documented to provide minor protection IFN-γ is an important cytokine involved in the against coccidiosis. However, humoral immunity may Th1-mediated immune response. Chicken IFN-γ could also contribute to the formation of protective immune elicit lymphocytes and enhance expression of MHC class responses [35]. Furthermore, Wallach [36] pointed out II antigens [38]. IFN-γ could also reduce sporozoites de- that antibodies could inhibit parasite development and velopment without affecting the sporozoite invasion of provide passive immune protection. Lin et al. [37] re- host cells [39]. In previous research, higher titers of ported that birds immunised with the E. tenella rEF-1α IFN-γ were detected in the EmMIC7 vaccinated birds [20]. In this study, higher IFN-γ titers in the vaccinated birds were also detected than those in the control birds. These results demonstrate that EmSAG could elicit Th1 cellular immune responses against E. maxima. It has been noted that cell-mediated immunity is the most important immune response to Eimeria infection. + + In this study, the CD4 and CD8 percentages were higher in the groups immunised with pVAX1-EmSAG and rEmSAG protein, when compared to the control groups. This demonstrated that EmSAG might be able to stimulate cellular immunity. IL-4 is known as a marker of the Th2 immune re- sponse [40] and has been reported as an important fac- tor in protective immunity against parasite infections [41]. Tian et al. [42] reported that groups vaccinated with EmGAPDH exhibited higher concentrations of IL-4 compared to control groups injected with PBS and pVAX1 alone. The results of this study demonstrated an Fig. 6 Levels of EmSAG-specific IgY/IgG in chicken sera were increased IL-4 level in the EmSAG-vaccinated birds measured using ELISA. The titers of the EmSAG-specific IgY/IgG are compared to those in the negative control. Coupled with expressed as the mean + SD the high antibody concentration, these data indicate that Liu et al. Parasites & Vectors (2018) 11:325 Page 8 of 12 Fig. 7 Levels of cytokines IL-4 (a), IL-17 (b), IFN-γ (c) and TGF-β (d) in chicken sera were measured using ELISA. Bars with different letters are significantly different (P < 0.05) + + + + Fig. 8 T lymphocytes subpopulations were detected by the flow cytometry technique. a CD4 T lymphocytes (CD3 CD4 , region Q2). b CD8 T + + lymphocytes (CD3 CD8 , region Q2) Liu et al. Parasites & Vectors (2018) 11:325 Page 9 of 12 Table 1 Flow cytometry analysis of the percentages of T lymphocyte subsets (mean ± SD, %) Marker Group Pre-immunized (%) 1st immunized (%) 2nd immunized (%) + a a a CD4 PBS 23.74 ± 3.21 24.18 ± 1.94 24.68 ± 3.58 a a a pVAX1.0 control 24.28 ± 3.67 25.26 ± 3.55 25.12 ± 2.84 a a a pET-32a(+) control 24.04 ± 3.39 23.32 ± 2.88 24.50 ± 2.13 a a b rEmSAG 23.12 ± 3.4 28.14 ± 4.67 35.70 ± 2.94 a b b pVAX-EmSAG 24.96 ± 1.66 35.24 ± 3.51 40.92 ± 7.66 + a a a CD8 PBS 34.74 ± 3.13 34.18 ± 3.94 35.16 ± 1.94 a a a pVAX1.0 control 34.64 ± 1.35 35.26 ± 3.65 35.08 ± 2.89 a a a pET-32a(+) control 35.32 ± 2.57 34.20 ± 5.37 34.84 ± 3.72 a b b rEmSAG 34.8 ± 6.19 45.44 ± 3.83 57.02 ± 1.85 a b c pVAX-EmSAG 34.28 ± 5.61 49.04 ± 2.76 64.28 ± 3.65 Note: In each column, different letters indicate a significant difference (P < 0.05) between numbers. There is no significant difference (P > 0.05) between numbers with the same letter EmSAG could stimulate humoral immune response to vaccinated with the rEmSAG protein and E. maxima. pVAX1-EmSAG showed higher concentrations of A new class of T-helper cells known as Th17 cells is TGF-β1 than that of control groups. However, the exact associated with interleukin IL-17 production [43]. In the function of TGF-β in protecting against coccidiosis avian immune system, IL-17 functions as a stimulator of needs further investigation. cytokine productions [44]. It has been confirmed that Antibodies and cytokines have been shown to influ- co-vaccination of IL-17 with 3-1 E protein induced bet- ence the protective immunity against coccidiosis infec- ter protection against E. acervulina than 3-1 E alone [4]. tions. In previous reports, monoclonal antibodies Previously, it was reported that the immunisation of ani- showed the ability to reduce oocyst shedding and pro- mals with DNA vaccines produced higher levels of IL-17 vide partial protection against E. maxima or E. tenella production [45]. However, IL-17 neutralised antibody challenge infections [52, 53]. IL-4 could enhance the treated birds showed enhanced IL-12 and IFN-γ expres- production of the antibody [54]. Chickens injected with sion [46]. In this research, a significant increment of IL-17 recombinant IFN-γ showed improved protective im- concentrations was detected ten days after the last im- munity following E. acervulina infection [55–57]. Rose munisation. This finding coupled with the high IFN-γ ti- et al. [58] found that neutralising IFN-γ though mono- ters, indicated that EmSAG could induce Th1 and Th17 clonal antibody could increase the output of oocysts and response. However, the exact function of TH17 in immun- loss of body weight. Additionally, oocyst shedding was isation against Eimeria spp. needs further investigation. decreased in birds co-injected with IFN-γ or TGF-β with TGF-β is a cytokine that has been recognised as part the 3-1E DNA vaccine compared to the birds inoculated of the immune suppression mechanism [47, 48]. TGF-β with the DNA vaccine alone [59]. Lillehoj et al. [60] re- has been reported to induce protective immunity and in- ported that co-vaccination with EtMIC2 and TGF-β sig- creased TNF-α production [49, 50]. Hoan et al. [51] also nificantly reduced oocyst shedding and enhanced weight reported that EbAMA1 could induce significantly higher gains beyond those injected by EtMIC2 alone. Zhang et concentrations of TGF-β1 and IL17 in the vaccinated al. [46] found that the IL-17 neutralised birds showed groups. Likewise, in the current research, birds decreased fecal oocyst output and caecal lesion scores, Table 2 Effects of SAG against E. maxima challenge on different parameters Group Average body weight gain (g) (mean ± SD) Mean lesion scores (mean ± SD) Oocyst decrease ratio (%) Anti-coccidial index a a a Unchallenged control 55.75 ± 18.29 0.00 ± 0.00 100 200 b b b Challenged control 23.84 ± 14.52 2.16 ± 0.13 0 81.12 b b b pVAX1 control 25.16 ± 12.86 2.09 ± 0.18 2.76 84.18 b b b pET 32a control 24.93 ± 14.32 2.13 ± 0.14 2.54 83.36 c c c rEmSAG 48.12 ± 16.10 1.22 ± 0.10 75.93 173.07 c c c pVAX1-SAG 49.17 ± 14.82 1.13 ± 0.16 76.64 175.88 Note: In each column, different letters indicate a significant difference (P < 0.05) between numbers. There is no significant difference (P > 0.05) between numbers with the same letter Liu et al. Parasites & Vectors (2018) 11:325 Page 10 of 12 as well as increased body weight gain. Geriletu et al. [44] Authors’ contributions LXR directed the project and participated in all management co-ordination reported that vaccination with IL-17A and MZP5-7 re- regarding this study. LTQ wrote the manuscript and performed experiments. duced oocyst shedding and decreased intestinal lesions HJW, LYL, WS and ZZY helped to perform laboratory tests and analysed data. following E. tenella challenge compared to inoculation ME helped to rectify grammatical mistakes in manuscript. All analytical tools and reagents were provided by SXK, YRF and XLX. All authors read and with MZP5-7 alone. In this study, challenge experiments approved the final manuscript. showed that the concentration of anti-EmSAG anti- bodies, IFN-γ, IL-4, TGF-β and IL-17 were increased in Ethics approval This study was conducted in accordance with the recommendations of the both the rEmSAG protein and pVAX1-SAG immunised guidelines of the Animal Ethics Committee, Nanjing Agricultural University, groups. Additionally, the jejunal lesions, loss of body China. The protocol was approved by the Science and Technology Agency weight and oocyst production ratio were all decreased. of Jiangsu Province (approval ID, SYXK (SU) 2010–0005). These results indicate that the antibodies and cytokines Competing interests played a role in the immune protection induced by the The authors declare that they have no competing interests. rEmSAG protein. Localisation of the proteins is critical to understanding Publisher’sNote the role which they play in parasite binding and the inva- Springer Nature remains neutral with regard to jurisdictional claims in sion of the host cell [61, 62]. Previous studies reported published maps and institutional affiliations. that monoclonal antibodies were able to detect proteins Received: 15 January 2018 Accepted: 20 May 2018 on the parasite surface, such as EtSAG1 and the micro- nemes of the sporozoites and merozoites [63–65]. Jenkins et al. [66] showed the immune-mapped protein 1 could be References detected in the sporozoites. Zhang et al. [31]found 1. Shirley MW, Ivens A, Gruber A, Madeira AMBN, Wan K-L, Dear PH, et al. The Eimeria genome projects: a sequence of events. Trends Parasitol. 2004;20: EaMIC3 on the apical tip of E. acervulina sporozoites. 199–201. Our findings suggest that EmSAG is expressed in the 2. Wallach M, Smith NC, Braun R, Eckert J. Potential control of chicken sporozoite and merozoite stages of E. maxima, and might coccidiosis by maternal immunization. Parasitol Today. 1995;11:262–5. 3. Wallach MG, Ashash U, Michael A, Smith NC. Field application of a subunit play an important role in the host invasion mechanism. vaccine against an enteric protozoan disease. PLoS One. 2008;3:e3948. 4. Ding X, Lillehoj HS, Quiroz MA, Bevensee E, Lillehoj EP. Protective immunity against Eimeria acervulina following in ovo immunization with a Conclusions recombinant subunit vaccine and cytokine genes. Infect Immun. 2004;72: In conclusion, our findings indicate that vaccination with 6939–44. EmSAG is capable of eliciting both humoral immunity 5. Shah MAA, Yan R, Xu L, Song X, Li X. A recombinant DNA vaccine encoding Eimeria acervulina cSZ-2 induces immunity against experimental E. tenella and cell-mediated immunity, exploring a moderate pro- infection. Vet Parasitol. 2010;169:185–9. tective immunity against E. maxima. This work suggests 6. Blake DP, Tomley FM. Securing poultry production from the ever-present that EmSAG could be used as an effective vaccine candi- Eimeria challenge. Trends Parasitol. 2014;30:12–9. 7. Widera G, Austin M, Rabussay D, Goldbeck C, Barnett SW, Chen M, et al. date to resist E. maxima infection. Increased DNA vaccine delivery and immunogenicity by electroporation in vivo. J Immunol. 2000;164:4635–40. Abbreviations 8. Song X, Zhang Z, Liu C, Xu L, Yan R, Li X. Evaluation of the persistence, ACI: Anti-coccidial index; Cy3: Cyanine dyes 3; DAPI: 2-(4-amidinophenyl)-6- integration, histopathology and environmental release of DNA vaccine indolecarbamidine dihydrochloride; EmSAG: surface antigen gene of Eimeria encoding Eimeria tenella TA4 and chicken IL-2. Vet Parasitol. 2016;229:22–30. maxima; IFN-γ: Interferon-γ; IgG-HRP: horseradish peroxidase labeled 9. Chapman H. Milestones in avian coccidiosis research: a review. Poultry Sci. immunoglobulin G; IL-17: Interleukin-17; IL-4: Interleukin-4; IPTG: Isopropyl-B- 2014;93:501–11. D-thiogalactopyranoside; RT-PCR: Reverse transcription-polymerase polymer- 10. Dowd KA, Ko SY, Morabito KM, Yang ES, Pelc RS, Demaso CR, et al. Rapid ase chain reaction; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel development of a DNA vaccine for Zika virus. Science. 2016;354:237. electrophoresis; TGF-β1: Transforming growth factor-β1; Th1: helper T cell 1; 11. 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Molecular characterisation and the protective immunity evaluation of Eimeria maxima surface antigen gene

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Biomedicine; Parasitology; Entomology; Tropical Medicine; Infectious Diseases; Veterinary Medicine/Veterinary Science; Virology
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Abstract

Background: Coccidiosis is recognised as a major parasitic disease in chickens. Eimeria maxima is considered as a highly immunoprotective species within the Eimeria spp. family that infects chickens. In the present research, the surface antigen gene of E. maxima (EmSAG) was cloned, and the ability of EmSAG to stimulate protection against E. maxima was evaluated. Methods: Prokaryotic and eukaryotic plasmids expressing EmSAG were constructed. The EmSAG transcription and expression in vivo was performed based on the RT-PCR and immunoblot analysis. The expression of EmSAG in sporozoites and merozoites was detected through immunofluorescence analyses. The immune protection was assessed based on challenge experiments. Flow cytometry assays were used to determine the T cell subpopulations. The serum antibody and cytokine levels were evaluated by ELISA. Results: The open reading frame (ORF) of EmSAG gene contained 645 bp encoding 214 amino acid residues. The immunoblot and RT-PCR analyses indicated that the EmSAG gene were transcribed and expressed in vivo. The EmSAG proteins were expressed in sporozoite and merozoite stages of E. maxima by the immunofluorescence assay. Challenge experiments showed that both pVAX1-SAG and the recombinant EmSAG (rEmSAG) proteins were successful in alleviating jejunal lesions, decreasing loss of body weight and the oocyst ratio. Additionally, these + + experiments possessed anticoccidial indices (ACI) of more than 170. Higher percentages of CD4 and CD8 T cells were detected in both EmSAG-inoculated birds than those of the negative control groups (P < 0.05). The EmSAG- specific antibody concentrations of both the rEmSAG and pVAX1-EmSAG groups were much higher than those of the negative controls (P < 0.05). Higher concentrations of IL-4, IFN-γ, TGF-β1 and IL-17 were observed more in both the rEmSAG protein and pVAX1-SAG inoculated groups than those of negative controls (P < 0.05). Conclusions: Our findings suggest that EmSAG is capable of eliciting a moderate immune protection and could be used as an effective vaccine candidate against E. maxima. Keywords: Eimeria maxima, Surface antigen, Cytokines, Vaccine, Immunity * Correspondence: lixiangrui@njau.edu.cn College of Veterinary Medicine, Nanjing Agriculture University, 1 Weigang, Nanjing, Jiangsu 210095, People’s Republic of China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. Parasites & Vectors (2018) 11:325 Page 2 of 12 Background (Invitrogen Biotech, Shanghai, China) and verification, the Coccidiosis is recognised as a major parasitic disease in positive clones were confirmed as pET-32a/ EmSAG. chickens seriously affecting the efficiency of feed conver- sion and leading to decreased production. Eimeria Expression of the recombinant EmSAG protein maxima has been recognised as one of the most eco- The sequence identity of EmSAG was compared to the nomically significant species of Eimeria [1]. Currently, known SAG sequences of other Eimeria spp. and prophylactic chemotherapy with anticoccidial drugs is assessed using the BLASTx and BLASTp search tools the major control strategy for coccidiosis. Traditional (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The amino acid anticoccidial drugs and live vaccines have their own de- sequence of EmSAG was used to predict N-terminal sig- fect [2]. Subunit vaccines encoding the Eimeria proteins nal peptides through a bioinformatics online program which stimulated protective immunity were accepted as (http://www.cbs.dtu.dk/services/SignalP/). The clado- effective vaccines against coccidiosis [3–5]. Recently, gram was made using the MEGA 6.0 programme with many reports have shown that cell-mediated immunity the neighbour-joining method. The pET-32a/EmSAG could be stimulated by DNA vaccines [6–10]. was expressed in E. coli BL21 (DE3) as described previ- Surface antigens have been proven to confer protec- ously [21]. The recombinant protein was purified and tion against coccidiosis by altering key processes in host the concentration of the sample was determined using cell invasions [11]. The SAGs protein of Eimeria tenella the Bradford method [22]. The rEmSAG protein was is capable of inducing an immune response against coc- kept frozen (-70 °C) until further analysis. cidiosis in chickens [12]. Therefore, surface antigens and cell adhesion proteins have been suggested as promising Development of anti-EmSAG antibodies against the vaccine candidates against parasitic infections [13, 14]. rEmSAG protein Eimeria maxima is regarded as a highly immunopro- Rat polyclonal anti-EmSAG antibodies were generated tective species within the family of Eimeria spp. affecting in the Sprague-Dawley rats at 4 weeks of age. Rats were chickens [15–19]. In this study, subunit and DNA vac- subcutaneously immunised with a total of 0.3 mg of cines made from EmSAG were evaluated for their pro- rEmSAG protein mixed with Freund’s complete adju- tection against E. maxima. vant. Fourteen days after the first immunisation, the rats were given a booster injection with 0.3 mg of rEmSAG Methods protein in Freund’s incomplete adjuvant. Three booster Chickens and parasites doses were given at 1-week intervals. Finally, rat serum Eimeria-free birds at one day of age were reared in cap- containing antibodies were obtained after the last tivity with provided water and feed ad libitum. The birds booster injection and kept frozen (at -70 °C) until subse- were placed in a coccidia-free environment. The Jiangsu quent analysis. Pre-immunisation serum was obtained strain of E. maxima was developed and maintained in for later use as the negative control [23]. Eimeria-free birds our laboratory. Sporozoites from E. maxima oocysts were cleaned and sporulated as previ- Construction of eukaryotic plasmid of EmSAG ously described [20]. The construction of eukaryotic plasmid of EmSAG was conducted and purified as previously described [20]. Amplification and prokaryotic expression of EmSAG Briefly, the EmSAG fragment was inserted into the The construction of prokaryotic expression of EmSAG pVAX1, following the sequence analysis (Invitrogen Bio- was conducted as previously described [20]. Briefly, the tech) and verification, the positive clones were con- EmSAG-encoding sequence (GenBank: XM_013482011.1) firmed as pVAX1-EmSAG. The plasmids encoding was amplified by PCR. EmSAG-specific primers were uti- EmSAG were extracted using EndoFree Plasmid MEGA lised for the PCR assays: SAG1 (forward primer: 5'-CGC Kit (Qiagen, Valencia, CA, USA). The concentration of GGA TCC GAC ACA ATC TCC AGC CCT-3'; BamHI the sample was determined using as per the method sug- restriction sites underlined) and SAG2 (reverse primer: gested earlier [20]. Finally, the plasmids were kept frozen 5'-ATT GCG GCC GCT CAA ATG AGA ACA GAT (-20 °C) until subsequent analysis. GCG-3'; NotI restriction sites underlined) with E. maxima cDNA as a template. The amplification products of Immunoblot analysis of native EmSAG and rEmSAG proteins EmSAG cloned in pMD-19T (TaKaRa, Dalian, China) re- Immunoblot analyses for rEmSAG and native EmSAG sulted in the formation of recombinant plasmid were performed as described in an earlier work [20]. Rat pMD-19T-EmSAG. Subsequently, the EmSAG gene was anti-rEmSAG sera (dilutions of 1:200) were used to de- inserted into the pET-32a (+) (Novagen, Madison, WI, tect sporozoites. Chicken anti-E. maxima sera (dilutions USA) frame of expression vector system and confirmed by of 1:100) were used to detect the rEmSAG protein. Goat endonuclease digestion. Following sequence analysis anti-rat HRP-IgG and donkey anti-chick HRP-IgG Liu et al. Parasites & Vectors (2018) 11:325 Page 3 of 12 (Sigma-Aldrich, St. Louis, MO, USA) were used as a sec- Assessment of immune protection ondary antibody. The chickens were monitored for body weight gain and signs of immune protection (jejunal lesion score, survival rate and change in oocyst ratio). Lesion scrapings were Transcription and expression of pVAX1-EmSAG in vivo microscopically examined for any coccidia, whenever The EmSAG transcription in vivo was performed based there was doubt of truly coccidia-induced lesions. The je- on the RT-PCR and immunoblot analysis, as previously junal lesion scores of the birds were also evaluated, as de- described [24]. Briefly, in coccidia-free chickens, a total scribed in previous research [23]. The body weight gains of 100 μg pVAX1-EmSAG plasmid was intramuscularly were measured at different time-points: the days of vac- injected into the legs. In the pVAX1 control group, the cination, at the time of the coccidia challenge, and at the 100 μg pVAX1 plasmid was injected into the legs. One end of the test. All the jejunal contents from each bird week post-immunisation, the tissues from vaccinated were harvested and used to evaluate the oocyst counts as and non-vaccinated chickens were collected for both described in a previous study [26]. Using the McMaster’s RT-PCR and immunoblot analyses. EmSAG-specific counting method, oocysts and the oocyst ratio were primers were utilised for the RT-PCR assays. Rat assessed as previously described [27]. Anticoccidial index anti-rEmSAG sera (dilutions of 1:200) were used to de- (ACI) values were evaluated as per the standard formula tect pVAX1-EmSAG expression. The secondary antibody for assessing protection against E. maxima [20]. was HRP-conjugated goat anti-rat IgG (Sigma-Aldrich). ELISA analysis of the serum antibody and cytokine Location of the EmSAG protein in sporozoites and EmSAG-specific IgY/IgG antibodies were detected by merozoites stages ELISA using the rEmSAG protein as a coating antigen, Immunofluorescence technique was used to locate following previous protocols [28]. The serum samples EmSAG in sporozoites and merozoites as previously de- (1:50 dilution) were detected using the secondary anti- scribed [25]. Nuclei were probed with 2-(4-amidinophe- bodies of donkey anti-chicken HRP-conjugated IgG nyl)-6-indole carbamidinedihydrochloride (DAPI, monoclonal antibody (Sigma-Aldrich). The experiment Sigma-Aldrich). Rat anti-EmSAG sera (1:100 dilutions) was completed in duplicate. were used as the primary antibody. The secondary anti- For cytokines level analysis, serum samples were ob- body was Cy3-conjugated goat anti-rat IgG (Beyotime, tained and measured as previously described [29]. Briefly, Haimen, Jiangsu, China) (dilution of 1:1000). The slides 10 days after the last inoculation, the serum samples of the were analysed using fluorescence microscopy (Nikon, birds (n = 5) per group were harvested to evaluate the cy- Tokyo, Japan). tokines. The titers of IL-4, IL-17, IFN-γ and TGF-β1were measured using ELISA kits (CUSABIO, Wuhan, China). Experimental design The data was pooled from three independent experiments. Chickens at 14 days of age, negative for Eimeria were placed in six groups, each including 30 birds. The chick- Determination of T-cell response ens were inoculated intramuscularly injection with the The counts of T cells in the treatment groups were eval- pVAX1-SAG (100 μg/chick) or rEmSAG protein (200 uated by flow cytometry analysis as previously described μg/chick). In the pVAX1 control chickens, a total of 100 [30, 31]. Spleens were extracted from 5 chickens of each μg pVAX1 plasmid was injected into the legs. In the group at pre-, first-, and second-vaccination days. Lym- pET-32a control chickens group, a total of 200 μg phocytes were obtained from the spleens were stained pET-32a protein was injected as above. The challenged with SPRD-conjugated CD3 monoclonal antibodies. The and unchallenged control birds were immunised with cells were then probed with PE-conjugated mouse PBS. One week later, the birds were boosted with the monoclonal anti-chicken CD4 or the PE-conjugated same route as the primary immunisation. Subsequently, mouse monoclonal anti-chicken CD8 (Southern Biotech- 7 days after the last immunisation, 1 × 10 sporulated nology Associates, Birmingham, AL, USA). Using FACS oocysts of E. maxima were given to all the birds except flow cytometer, the stained cells were analysed with Cell the negative control birds. Seven days later, the birds Quest software (BD Biosciences, San Jose, CA, USA). were euthanised to measure their immune response and degree of coccidial protection. Moreover, the birds Statistical analysis (n = 5 per group) were placed in another coccidia-free All data was expressed as the mean ± standard deviation room. Finally, 10 days after the last immunisation, the using the SPSS Statistical Software (SPSS Inc., Chicago, serum samples were harvested and kept frozen (-20 °C) IL, USA). The data were analysed with one-way ANOVA until further antibodies and cytokine production ana- using Duncan’s post-hoc test and considered to be statis- lysis could be conducted. tically significant at P < 0.05. Liu et al. Parasites & Vectors (2018) 11:325 Page 4 of 12 Results EmSAG sequence analysis Using E. maxima cDNA as a template, the PCR product of EmSAG was isolated and ligated with pMD19-T. Se- quence analysis showed that the EmSAG ORF encoded a protein of 24.73 kDa with a pI of 4.808. As shown in Fig. 1, the phylogenetic tree formulation indicated that the kinship of EmSAG protein was highly related to the EtTA4 and EnNA4 when compared with other Eimeria spp. (E. mitis, E. brunetti, E. praecox, E. acervulina and E. necatrix). The amino acid sequence was analysed with the SignalP programme. The findings suggested an obvi- ous signal peptide possessed a cleavage site between pos- ition 21 and 22. Purification of the rEmSAG protein The pET32a/EmSAG plasmids were expressed in E. coli BL21. After IPTG induction, the rEmSAG proteins were harvested. The purified fusion rEmSAG protein was ap- proximately 43 kDa (Fig. 2). This calculated total value of 43 kDa was considered accurate as the sum of both the approximate 20 kDa length of pET-32a (+) and the approximate 23 kDa length of the EmSAG protein. Immunoblot analysis of native and rEmSAG proteins The native and rEmSAG proteins were evaluated by the western blot method (Fig. 3). The rEmSAG protein was tested using chicken E. maxima-specific antibodies, but not by the antibodies of unimmunised chickens. Further- more, the western blot assay also showed a band of al- most 26 kDa belonging to the sporozoites protein detected by rat anti-rEmSAG antibodies (Fig. 3), in con- trast to the serum from the negative control rats that did not display any bands. Fig. 2 Purified rEmSAG protein separated by SDS-PAGE. Lane M: pre-stained protein marker; Lane 1: the purified rEmSAG protein Identification of EmSAG location in sporozoites and stained with Coomassie brilliant blue merozoites The location of EmSAG in sporozoites and merozoites of E. maxima was confirmed using immunofluorescence analyses (Fig. 4). The EmSAG protein was detected using rat anti-rEmSAG antibodies, and Cy3-conjugated goat anti-rat IgG as secondary antibodies shown in red Fig. 1 The phylogenetic tree was constructed using CLUSTAL W alignment and neighbour-joining method of the software MEGA 6.0 Liu et al. Parasites & Vectors (2018) 11:325 Page 5 of 12 Fig. 3 Immunoblot analysis for native and rEmSAG proteins. Lane M: pre-stained protein marker; Lane 1: rEmSAG recognised by chick anti-E. maxima serum; Lane 2: rEmSAG protein tested against unimmunised chicken sera; Lane 3: E. maxima protein from sporozoites detected by rat anti-rEmSAG sera; Lane 4: protein of E. maxima sporozoites detected by unimmunised rat sera fluorescence, whereas no red fluorescence was detected in Analysis of T cell subpopulations cells probed with the pre-immune rat serum. The nuclei To evaluate the EmSAG specific T-cell responses, flow of the sporozoites and merozoites were visualised as blue. cytometry assays were used to analyse the CD4 and These results suggest that EmSAG was expressed in both CD8 T cells. The spleen lymphocytes were collected at the sporozoite and merozoite stages of E. maxima. pre-, first-, and second-inoculation time-points (Fig. 8). After the last vaccination, the percentage of CD4 in the EmSAG-immunised chickens was higher (ANOVA, Identification of transcription and expression of F = 46.28, P < 0.0001), than those in the PBS group, (4, 20) pVAX1-EmSAG in vivo pVAX1.0 group, and the pET-32a (+) group. Regarding Transcription and expression of pVAX1-SAG in vivo was CD8 T cells, EmSAG groups showed a higher evaluated through RT-PCR, using the EmSAG-specific (ANOVA, F = 43.59, P < 0.0001) percentage, (4, 20) primers. A specific DNA band was detected belonging to whereas the PBS, pVAX1 and pET-32a (+) control pVAX1-EmSAG in the tissues of the injected site (Fig. 5a, group remained at low levels after the second immun- Lane 4). The RNA samples from non-inoculated and isation (Table 1 and Fig. 8). pVAX1-inoculated tissues did not detect any band in the RT-PCR analyses (Fig. 5a, Lanes 1, 2 and 3). In addition, expression of pVAX1-SAG in vivo was de- Immune protection of EmSAG against E. maxima tected through immunoblot analysis. A unique band of To analyse the immune protection of EmSAG against E. approximately 26 kDa was detected in the maxima, the challenge experiments were assessed. The de- pVAX1-EmSAG-vaccinated muscle sample. In contrast, grees of immune protection conferred by vaccinations of no band was shown in the pVAX1-immunised muscle pVAX1-EmSAG and rEmSAG proteins were measured, samples (Fig. 5b). These results indicate the successful and the results of ACI are described in Table 2.Birds inoc- transcription and expression of the EmSAG gene in vivo. ulated with EmSAG exhibited higher weight gains (ANOVA, F = 27.67, P < 0.0001) and greater de- (5, 174) creases in oocyst ratios when compared to all other groups. Determination of IgY/IgG and cytokines levels using ELISA The ACIs of the EmSAG-immunised chickens were more To evaluate the titers of IgY/IgG and the cytokines, than 170, providing moderate protective immunity. serum samples from the immunised birds (n = 5 per group) were harvested at 10 days after the last vaccin- ation. The anti-EmSAG IgY/IgG titers of each group are Discussion shown in Fig. 6. The IgY/IgG titers of both In this research, both DNA and recombinant protein EmSAG-immunised groups were much higher (ANOVA, vaccines encoding EmSAG of E. maxima were compared F = 77.78, P < 0.0001) compared to the controls. regarding their abilities to induce protection against E. (4, 20) The titers of cytokines were measured using ELISA maxima infection. These results indicated that inocula- (Fig. 7). The serum samples in both pVAX1-EmSAG tion with EmSAG could promote IgG levels in the sera and rEmSAG-immunised chickens displayed higher ti- and upregulated the titers of IL-4, IFN-γ, IL-17 and ters of IFN-γ (ANOVA, F = 43.59, P < 0.0001), TGF-β1. Furthermore, the data from the animal experi- (4, 20) IL-17 (ANOVA, F = 42.25, P < 0.0001), IL-4 ments proved that EmSAG-immunised groups could (4, 20) (ANOVA, F = 3.25, P = 0.033) and TGF-β1 produce ACIs of more than 170. Taken together, these (4, 20) (ANOVA, F = 48.12, P < 0.0001) compared to the data demonstrate that EmSAG vaccines could stimulate (4, 20) negative controls. moderate protection against E. maxima. Liu et al. Parasites & Vectors (2018) 11:325 Page 6 of 12 Fig. 4 Expression of EmSAG protein in sporozoites and merozoites at 100× magnification. a The sporozoites were detected by rat anti-rEmSAG antibodies. a1 Sporozoites were dyed by Cy3. a2 The nuclei were probed by DAPI. a3 Overlaps of Cy3 and DAPI. b The sporozoites were detected by unimmunised rat antibodies. b1 Cy3 stains. b2 DAPI stains. b3 Merge. c Merozoites were detected by rat anti-rEmSAG antibodies. c1 Cy3 stains. c2 DAPI stains. c3 Merge. d The merozoites were detected by unimmunised rat antibodies. d1 Cy3 stains. d2 DAPI stains. d3 Merge. Scale-bars:10 μm DNA and recombinant protein vaccines were reported challenged with E. maxima when compared with to induce immuno-protection to live parasite challenge. non-vaccinated and parasite-challenged groups. Xu et al. Higher body weight gain, lower fecal oocyst shedding [33] determined that pcDNA3.0-TA4-IL-2 could de- and reduced intestinal pathology were detected for im- crease caecal lesions and body weight loss as well as pro- mune protection. Jang et al. [32] reported that birds had duce an ACI of 192. Song et al. [34] reported that lower oocyst concentration in droppings and reduced in- chickens immunised with pMP13 plasmid showed sig- testinal pathology after vaccination with Gam82 and nificantly lower number of oocysts following the Liu et al. Parasites & Vectors (2018) 11:325 Page 7 of 12 Fig. 5 Expression and transcription of pVAX1-SAG in vivo were identified through RT-PCR and western blot assays. a RT-PCR of pVAX1-EmSAG transcription in chicken muscle. Lane M: DNA marker DL2000; Lanes 1 and 2: the muscle RNA sample from the non-inoculated chicken; Lane 3: the muscle RNA sample from the pVAX1-inoculated chicken; Lane 4: the muscle RNA sample from the pVAX1-EmSAG injected chicken. b Western blot of pVAX1-EmSAG in chicken muscle. Lane M: pre-stained protein marker; Lane 1: the protein sample from pVAX1-inoculated chickens; Lane 2: the protein sample from pVAX1- EmSAG inoculated chickens challenge with E. acervulina compared to those in the protein exhibited higher specific antibodies concentra- negative controls. Similar results were detected in this tion than the negative controls. In this research, the anti- study, both pVAX1-EmSAG and rEmSAG vaccines were body titers of the EmSAG-immunised animals were successful in alleviating jejunal lesions, decreasing loss higher than the negative controls. The findings of this of body weight and the oocyst ratio. investigation confirmed that EmSAG could induce The chick-anti-Eimeria specific antibodies have been humoral immune response. previously documented to provide minor protection IFN-γ is an important cytokine involved in the against coccidiosis. However, humoral immunity may Th1-mediated immune response. Chicken IFN-γ could also contribute to the formation of protective immune elicit lymphocytes and enhance expression of MHC class responses [35]. Furthermore, Wallach [36] pointed out II antigens [38]. IFN-γ could also reduce sporozoites de- that antibodies could inhibit parasite development and velopment without affecting the sporozoite invasion of provide passive immune protection. Lin et al. [37] re- host cells [39]. In previous research, higher titers of ported that birds immunised with the E. tenella rEF-1α IFN-γ were detected in the EmMIC7 vaccinated birds [20]. In this study, higher IFN-γ titers in the vaccinated birds were also detected than those in the control birds. These results demonstrate that EmSAG could elicit Th1 cellular immune responses against E. maxima. It has been noted that cell-mediated immunity is the most important immune response to Eimeria infection. + + In this study, the CD4 and CD8 percentages were higher in the groups immunised with pVAX1-EmSAG and rEmSAG protein, when compared to the control groups. This demonstrated that EmSAG might be able to stimulate cellular immunity. IL-4 is known as a marker of the Th2 immune re- sponse [40] and has been reported as an important fac- tor in protective immunity against parasite infections [41]. Tian et al. [42] reported that groups vaccinated with EmGAPDH exhibited higher concentrations of IL-4 compared to control groups injected with PBS and pVAX1 alone. The results of this study demonstrated an Fig. 6 Levels of EmSAG-specific IgY/IgG in chicken sera were increased IL-4 level in the EmSAG-vaccinated birds measured using ELISA. The titers of the EmSAG-specific IgY/IgG are compared to those in the negative control. Coupled with expressed as the mean + SD the high antibody concentration, these data indicate that Liu et al. Parasites & Vectors (2018) 11:325 Page 8 of 12 Fig. 7 Levels of cytokines IL-4 (a), IL-17 (b), IFN-γ (c) and TGF-β (d) in chicken sera were measured using ELISA. Bars with different letters are significantly different (P < 0.05) + + + + Fig. 8 T lymphocytes subpopulations were detected by the flow cytometry technique. a CD4 T lymphocytes (CD3 CD4 , region Q2). b CD8 T + + lymphocytes (CD3 CD8 , region Q2) Liu et al. Parasites & Vectors (2018) 11:325 Page 9 of 12 Table 1 Flow cytometry analysis of the percentages of T lymphocyte subsets (mean ± SD, %) Marker Group Pre-immunized (%) 1st immunized (%) 2nd immunized (%) + a a a CD4 PBS 23.74 ± 3.21 24.18 ± 1.94 24.68 ± 3.58 a a a pVAX1.0 control 24.28 ± 3.67 25.26 ± 3.55 25.12 ± 2.84 a a a pET-32a(+) control 24.04 ± 3.39 23.32 ± 2.88 24.50 ± 2.13 a a b rEmSAG 23.12 ± 3.4 28.14 ± 4.67 35.70 ± 2.94 a b b pVAX-EmSAG 24.96 ± 1.66 35.24 ± 3.51 40.92 ± 7.66 + a a a CD8 PBS 34.74 ± 3.13 34.18 ± 3.94 35.16 ± 1.94 a a a pVAX1.0 control 34.64 ± 1.35 35.26 ± 3.65 35.08 ± 2.89 a a a pET-32a(+) control 35.32 ± 2.57 34.20 ± 5.37 34.84 ± 3.72 a b b rEmSAG 34.8 ± 6.19 45.44 ± 3.83 57.02 ± 1.85 a b c pVAX-EmSAG 34.28 ± 5.61 49.04 ± 2.76 64.28 ± 3.65 Note: In each column, different letters indicate a significant difference (P < 0.05) between numbers. There is no significant difference (P > 0.05) between numbers with the same letter EmSAG could stimulate humoral immune response to vaccinated with the rEmSAG protein and E. maxima. pVAX1-EmSAG showed higher concentrations of A new class of T-helper cells known as Th17 cells is TGF-β1 than that of control groups. However, the exact associated with interleukin IL-17 production [43]. In the function of TGF-β in protecting against coccidiosis avian immune system, IL-17 functions as a stimulator of needs further investigation. cytokine productions [44]. It has been confirmed that Antibodies and cytokines have been shown to influ- co-vaccination of IL-17 with 3-1 E protein induced bet- ence the protective immunity against coccidiosis infec- ter protection against E. acervulina than 3-1 E alone [4]. tions. In previous reports, monoclonal antibodies Previously, it was reported that the immunisation of ani- showed the ability to reduce oocyst shedding and pro- mals with DNA vaccines produced higher levels of IL-17 vide partial protection against E. maxima or E. tenella production [45]. However, IL-17 neutralised antibody challenge infections [52, 53]. IL-4 could enhance the treated birds showed enhanced IL-12 and IFN-γ expres- production of the antibody [54]. Chickens injected with sion [46]. In this research, a significant increment of IL-17 recombinant IFN-γ showed improved protective im- concentrations was detected ten days after the last im- munity following E. acervulina infection [55–57]. Rose munisation. This finding coupled with the high IFN-γ ti- et al. [58] found that neutralising IFN-γ though mono- ters, indicated that EmSAG could induce Th1 and Th17 clonal antibody could increase the output of oocysts and response. However, the exact function of TH17 in immun- loss of body weight. Additionally, oocyst shedding was isation against Eimeria spp. needs further investigation. decreased in birds co-injected with IFN-γ or TGF-β with TGF-β is a cytokine that has been recognised as part the 3-1E DNA vaccine compared to the birds inoculated of the immune suppression mechanism [47, 48]. TGF-β with the DNA vaccine alone [59]. Lillehoj et al. [60] re- has been reported to induce protective immunity and in- ported that co-vaccination with EtMIC2 and TGF-β sig- creased TNF-α production [49, 50]. Hoan et al. [51] also nificantly reduced oocyst shedding and enhanced weight reported that EbAMA1 could induce significantly higher gains beyond those injected by EtMIC2 alone. Zhang et concentrations of TGF-β1 and IL17 in the vaccinated al. [46] found that the IL-17 neutralised birds showed groups. Likewise, in the current research, birds decreased fecal oocyst output and caecal lesion scores, Table 2 Effects of SAG against E. maxima challenge on different parameters Group Average body weight gain (g) (mean ± SD) Mean lesion scores (mean ± SD) Oocyst decrease ratio (%) Anti-coccidial index a a a Unchallenged control 55.75 ± 18.29 0.00 ± 0.00 100 200 b b b Challenged control 23.84 ± 14.52 2.16 ± 0.13 0 81.12 b b b pVAX1 control 25.16 ± 12.86 2.09 ± 0.18 2.76 84.18 b b b pET 32a control 24.93 ± 14.32 2.13 ± 0.14 2.54 83.36 c c c rEmSAG 48.12 ± 16.10 1.22 ± 0.10 75.93 173.07 c c c pVAX1-SAG 49.17 ± 14.82 1.13 ± 0.16 76.64 175.88 Note: In each column, different letters indicate a significant difference (P < 0.05) between numbers. There is no significant difference (P > 0.05) between numbers with the same letter Liu et al. Parasites & Vectors (2018) 11:325 Page 10 of 12 as well as increased body weight gain. Geriletu et al. [44] Authors’ contributions LXR directed the project and participated in all management co-ordination reported that vaccination with IL-17A and MZP5-7 re- regarding this study. LTQ wrote the manuscript and performed experiments. duced oocyst shedding and decreased intestinal lesions HJW, LYL, WS and ZZY helped to perform laboratory tests and analysed data. following E. tenella challenge compared to inoculation ME helped to rectify grammatical mistakes in manuscript. All analytical tools and reagents were provided by SXK, YRF and XLX. All authors read and with MZP5-7 alone. In this study, challenge experiments approved the final manuscript. showed that the concentration of anti-EmSAG anti- bodies, IFN-γ, IL-4, TGF-β and IL-17 were increased in Ethics approval This study was conducted in accordance with the recommendations of the both the rEmSAG protein and pVAX1-SAG immunised guidelines of the Animal Ethics Committee, Nanjing Agricultural University, groups. Additionally, the jejunal lesions, loss of body China. The protocol was approved by the Science and Technology Agency weight and oocyst production ratio were all decreased. of Jiangsu Province (approval ID, SYXK (SU) 2010–0005). These results indicate that the antibodies and cytokines Competing interests played a role in the immune protection induced by the The authors declare that they have no competing interests. rEmSAG protein. Localisation of the proteins is critical to understanding Publisher’sNote the role which they play in parasite binding and the inva- Springer Nature remains neutral with regard to jurisdictional claims in sion of the host cell [61, 62]. Previous studies reported published maps and institutional affiliations. that monoclonal antibodies were able to detect proteins Received: 15 January 2018 Accepted: 20 May 2018 on the parasite surface, such as EtSAG1 and the micro- nemes of the sporozoites and merozoites [63–65]. Jenkins et al. 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Parasites & VectorsSpringer Journals

Published: May 30, 2018

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