Parasitol Res (2012) 110:2445–2453 DOI 10.1007/s00436-011-2784-7

ORIGINAL PAPER

Adjuvant effect of ginsenoside-based nanoparticles (ginsomes) on the recombinant vaccine against Eimeria tenella in chickens De-Fu Zhang & Hui Xu & Bing-Bing Sun & Jian-Qiu Li & Qian-Jin Zhou & Hong-Li Zhang & Ai-Fang Du

Received: 1 October 2011 / Accepted: 13 December 2011 / Published online: 4 January 2012 # Springer-Verlag 2011

Abstract An experiment was conducted to study the adjuvant effect of ginsomes on the recombinant profilin in coccidian-infected breeding birds. Three-day-old chickens were vaccinated with Eimeria tenella recombinant profilin antigen (10, 50, and 100 μg per chicken) with or without 50 μg ginsomes per chicken. The boost vaccination was carried out 14 days later. Two weeks after the booster, the chickens were challenged with 1.5×104 homologous sporulated oocysts. The specific antibody response, lymphocyte proliferation, and IL-1 release from lymphocyte were measured at 1–42 days after boost vaccination. Seven days postchallenge, the rate of survival, body weight gains (BWG) were examined then all chickens were sacrificed and lesion scores and oocysts per gram were monitored to evaluate the protective effects of the vaccination after challenge. Compared with the group of vaccinating with profilin only, groups of 50 and 100 μg antigen plus ginsomes significantly enhanced lymphocyte proliferation and IL-1 secretion. The profilin specific antibody level in the four vaccinated groups was significantly higher than in the control group and in groups vaccinated with profilin containing ginsomes than profilin only. In the groups vaccinated with profilin plus D.-F. Zhang : H. Xu : B.-B. Sun : Q.-J. Zhou : H.-L. Zhang : A.-F. Du (*) Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China e-mail: [email protected] H. Xu Shaoxing Entry-Exit Inspection and Quarantine Bureau, Shaoxing, Zhejiang 312000, China J.-Q. Li Zhejiang Gongshang University Hangzhou Commerce, Hangzhou, Zhejiang 310035, China

ginsomes, the BWG was significantly higher than that of group of profilin only, but there was no significant difference between profilin plus adjuvant ginsomes, diclazuril medicated and uninfected-unmedicated-unvaccinated control groups. The lesion scores in groups immunized with profilin plus ginsomes was significantly lower than that both of groups unimmunized-challenged-unmedicated control and group vaccinated with profilin only. Oocyst excretion in groups vaccinated with 50 or 100 μg profilin plus ginsomes was lower than that of groups vaccinated with profilin only. These results demonstrate that the adjuvant ginsomes can promote subunit vaccine to induce a strong immune response and protective effects.

Introduction Coccidiosis is a severe intestinal tract disease caused by apicomplexan intracellular protozoal parasite of the genus Eimeria. The high mortality, inefficient feed utilization, and the cost of medical prevention and treatment inflict the world poultry industry losses about US$ 3 billion annually (Dalloul and Lillehoj 2006; Michels et al. 2011; Shirley et al. 2004; Williams 1999). At present, prophylactic chemotherapy is still the main strategy of controlling coccidiosis. Vaccination is an alternative option for coccidiosis control (Dalloul and Lillehoj 2006; Michels et al. 2011; Jenkins et al. 2010; Yan et al. 2009). Compared with virulent or attenuated live vaccine, recombinant protein vaccine can induce good antibody response and has more efficiency to protect birds against challenge of Eimeria oocysts (Dalloul and Lillehoj 2006). Several Eimeria proteins have been studied as vaccine candidate (Allen and Fetterer 2002; Chapman et al. 2002; Lillehoj 1998; McDonald and Shirley 2009; Vermeulen et al. 2001; Williams 2002).

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Profilin antigen was identified from Eimeria acervulina merozoites firstly and encodes Eimeria 3-1E (subsequently identified as profilin), a conserved surface antigen of both sporozoites and merozoites of Eimeria tenella, Eimeria maxima, Eimeria acervulina, and Eimeria falciformis (Lillehoj et al. 2000; Fetterer et al. 2004; Ma et al. 2011; Jang et al. 2010; Zhao et al. 2011). Profilin contains an open reading frame encoding 170 residues of the amino acid from the carboxyl terminus of the E. tenella protein and expresses at Eimeria invasive stages, sporozoite and merozoite. Immunization with profilin recombinant protein or profilin DNA vaccine intranasal, intraocular, in vivo, or in ovo can induce cell-mediated immunity and protect chickens from infection of live Eimeria species (Lillehoj et al. 2000; Fetterer et al. 2004; Song et al. 2000; Min et al. 2002; Ding et al. 2004; Lillehoj et al. 2005; Ma et al. 2011; Lee et al. 2010b). The induced immunity can be boosted by adding of adjuvant (Ding et al. 2004; Lillehoj et al. 2000; Ma et al. 2011; Song and Hu 2009; Fetterer et al. 2004; Min et al. 2002; Song et al. 2000; Lillehoj et al. 2005; Lee et al. 2010b), which was regarded a promising vaccine candidate (Ma et al. 2011; Song et al. 2009a). Recent studies have demonstrated enhanced efficiency of recombinant vaccines in producing protective immunity by simultaneous injection of ginsenosides (Du et al. 2005). Ginsenosides are ginseng saponins, extracted from the root of traditional Chinese medicine Ginseng (GS), Panax ginseng C. A. Meyer. GS has been safely used as a tonic and haemostatic agent for more than 5,000 years in China (Song and Hu 2009) and can stimulate the natural resistance against infections (Yang et al. 2007). GS boosting of both cellular (Th1) and humoral (Th2) immune responses has been reported in various studies (Yang et al. 2007; Hu et al. 2003; Rivera et al. 2003). Song et al. (2009b) found coadministering 50 μg ginsomes with ovalbumin in ICR mice can significantly increase the levels of specific IgG titer, subclass levels, and production of IFN-γ and IL-5. Qu et al. (2011) reported that mice immunized with recombinant SAG1 with ginsenoside Rg1 had a more distinct immune response and survived longer than control mice when challenged with Toxoplasma gondii. Hu et al.(2003) reported that GS and Rd1 can induce a significantly increased lymphocyte proliferation and antibody production in dairy cattle immunized with Staphylococcus aureus bacterin. Yang et al. (2007) observed an enhanced splenocyte proliferation, IgG antibody titers, and promoted production of Th1 and Th2 cytokines and their up-regulated gene expression levels when immunizing mice with ovalbumin and ginsenoside Rd. The present study was designed to investigate the adjuvant effect of ginsenoside-based nanoparticles (ginsomes) on the immune responses induced by profilin recombinant vaccine and subsequent protection against E. tenella.

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Materials and methods Birds, parasites, and adjuvants Non-immune chickens were reared in clean brooder cages in hygienically controlled environment. The birds were provided with coccidiostat-free feed and water without any antibiotic. Birds were shifted to animal containment facility prior to challenge with virulent oocysts. All procedures related to the animals, and their care conformed to the internationally accepted principles as found in the Guidelines for Keeping Experimental Animals issued by the government of China. Ginsenoside-based nanoparticles (ginsomes) were kindly granted by Professor Songhua Hu (Institute of Traditional Chinese Veterinary Medicine, College of Animal Sciences, Zhejiang University). The E. tenella oocysts were stored in 2.5% potassium dichromate reagent in 4°C and propagated in 2-week-old chickens and purified before used. Recombinant E. tenella profilin protein Recombinant profilin fusion protein was produced in the pET-30a system (Novagen), and the resulting clones were sequenced. His fusion proteins were expressed in Escherichia coli BL21 (DE3). Protein expression of profilin antigen was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot using the mouse anti-His monoclonal antibody (Novagen) as the primary antibody. The membrane was stained by nitro blue tetrazolium substrate. The soluble recombinant profilin protein with 6× His-tag was purified by affinity chromatography with Ni-conjugated Sepharose (Sigma). The purity of protein was checked by SDS-PAGE, and the protein concentration was estimated by absorbance measurements at A280 nm. A stock protein was prepared and stored at −80°C until use. Immunization and sampling Experiment A One hundred non-immune chickens were randomly distributed into five groups (20 chickens each group). Chickens in group 1 were kept as unimmunized control and treated with 100 μl physiological saline solution. Chickens in group 2 were vaccinated subcutaneously in the breast region at 3 days of age with 100 μg recombinant profilin antigen. Chickens in groups 3–5 were vaccinated with 10, 50, and 100 μg recombinant profilin protein plus 50 μg ginsomes at 3 days of age, respectively. A booster was carried out 14 days later with the same amount as the first immunization. Blood samples were collected (four chickens each group) 1 week after the booster to

Parasitol Res (2012) 110:2445–2453

measure IgG level, blood lymphocyte proliferation, and IL1 inducement. The experimental protocol is illustrated as Fig. 1a. Experiment B Two hundred ten non-immune chickens were randomly divided into seven groups (groups A–G) of 30 individuals each. Chickens in group A, B, and G were kept as controls: the positive control (group A, unimmunized challenged and unmedicated control), drug-treated control (group B, unimmunized challenged and medicated with 1 mg diclazuril/kg feed), and the untreated control (group G, unimmunized unchallenged and unmedicated control). Chickens in group C were vaccinated subcutaneously at 3 days of age with 100 μg recombinant profilin antigen only. Chickens in group D–F were vaccinated subcutaneously with 10, 50, and 100 μg recombinant profilin antigen mixed with 50 μg ginsomes, respectively. The immunized birds in group C–F were boosted 2 weeks after the first immunization. The control chickens in group A, B, and G were injected with physiological saline solution in the same manner. All chickens, except for those in group G, were orally challenged with 1.5 × 10 4 E. tenella sporulated oocysts each bird 2 weeks after the booster and coccidial oocysts were surveyed during the fifth and seventh day postchallenge. Table 1 and Fig. 1b illustrated the experimental protocol.

Enzyme-linked immunosorbent assay Serum samples were analyzed for measurement of serum IgG by an indirect double antibody sandwich enzymelinked immunosorbent assay (ELISA) described before (Chiani et al. 2009). Briefly, 96-well microtiter plates were coated overnight at 4°C with 100 μl/well (10 μg/ml) recombinant profilin in carbonate buffer (pH 9.6). After three

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washes with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBST), the plates were blocked with blocking buffer (PBS containing 10% fetal calf serum) for 1 h at 37°C. After three washes with PBST, 100 μl serum sample per well was added and incubated for 1 h at 37°C. Individual sera were diluted 1:300. After three washes, plates were incubated for 2 h at 37°C with 100 μl/well of a 1:500 dilution of the peroxidase-conjugated secondary antibodies (rabbit anti-chick IgG antibody) in dilution buffer. After washing, peroxidase activity was detected by adding 100 μl/well orthophenylene diamine (Sigma; 0.4 mg/ml in 0.05 M phosphate citrate buffer, pH 5.0). After incubation for 15 min at 37°C, the reaction was stopped by addition of 50 μl 2 M sulfuric acid. An automated microtiter plate reader monitored reaction at 492 nm. Lymphocyte proliferation assay Peripheral blood lymphocyte was isolated by density gradient centrifugation over Ficoll-Hypaque (Amersham Biosciences), washed, diluted to 1 × 107 cells/ml in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS), 5 μg/ml concanavalin A (Con A, mitogen concanavalin A, Sigma) or profilin protein, 100 U/ml penicillin, and 100 μg/ml streptomycin in 96-well flat bottom plates at 37°C in a 5% CO2 incubator for 48 h; 10 μl 5 mg/ml freshly prepared 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) in PBS was added; the mixture was incubated for 4 h. After the supernatant was discarded, 100 μl formazan (Sigma) in dimethyl sulfoxide was added, and the plates were incubated at room temperature for 20 min. The supernatant was harvested for measurement of the optical density at 570 nm by an enzyme-linked immunosorbent assay microreader. IL-1 release from lymphocytes in vitro

Fig. 1 Schematic outlined the protocol of experiment A (a) and B (b). In experiment A, chickens were vaccinated (v) on day 3 and boosted (b) on day 17. From days 24 to 59, blood were sampled by cardiac puncture each week to detect IgG (g) (on days 24, 31, 38,45, 52, and 59), proliferation of peripheral blood lymphocytes (l), and IL-1 release (i) from lymphocytes (on days 31, 45, and 59). In experiment B, 210 chickens were weighed (w), randomly divided into seven groups and vaccinated (v) on day 3 and boosted (b) 2 weeks later. Chickens were orally challenged (c) with 1.5×104 homologous sporulated oocysts of E. tenella on day 31. On day 37, all chickens were weighed and killed to evaluate BWG, OPG (o), and lesion score of cecum (s)

Peripheral blood lymphocyte suspension supernatant stimulated by Con A was harvested. Cell suspensions were collected from the birds thymus under aseptic conditions and adjusted to a concentration of 1×107 cells/ml in RPMI 1640 supplemented with 10% FBS. To a 96-well flat-bottom microtiter plate, 100 μl of the cell suspension and equal volume of harvested supernatant solution were added. The plates were incubated at 37°C in 5% CO2 for 45 h; 10 μl of 5 mg/ml freshly prepared MTT in PBS was added. After the mixture was incubated at 37°C for 3 h, 50 μl 10%SDS– 0.01 mol HCl solution was added, and the plate was incubated at 37°C for 2 h and then kept at room temperature for 20 min. The optical density at 570 nm was measured by an enzyme-linked immunosorbent assay microreader.

2448 Table 1 Body weight (means ±SD grams, n030) before and at seventh day after challenged with E. tenella

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Groups

A B

Values with different superscripts in the same column differ significantly (P<0.05)

C D E F G

Treatment

Body weight

Unimmunized challenged unmedicated control Unimmunized challenged and diclazuril medicated 100 μg protein 10 μg protein+50 μg ginsomes 50 μg protein+50 μg ginsomes 100 μg protein+50 μg ginsomes Unimmunized unchallenged unmedicated control

Evaluation of immune protection To determine efficacy of vaccination, the survival rate, body weight gains (BWGs), lesion score, oocyst excretion, and protection rate were recorded. The survival rate was estimated by the number of surviving chickens divided by the number of initial chickens. The BWG of the chickens in each group was determined on 3 and 37 days of age (BWG0the body weight after challenge−body weight before challenge). The lesion score of chickens from each group was investigated according the method of Johnson and Reid (1970). Additionally, the complete feces of each group were collected separately, and oocysts were counted from 1 g of the feces using McMaster’s counting technique and the protection rate was calculated (Rose and Mockett 1983) as follows: (the number of oocysts from infectedunmedicated-unvaccinated control birds −the number of oocysts from vaccinated birds)/the number of oocysts from infected-unmedicated-unvaccinated control birds×100%.

Before challenge

After challenge

BWG

191.80±17.20a

244.06±17.51a

52.26a

190.78±11.05a

260.20±12.81bc

69.42bc

190.90±16.09a 196.70±17.81a 197.03±15.32a 196.70±11.72a 194.66±16.25a

249.09±21.20c 261.12±21.18b 261.50±19.36b 263.15±13.9b 265.50±26.09b

58.19c 64.42b 64.47b 66.45b 70.84b

beta-D-thiogalactopyranoside (0.5 mM) to express protein. The target protein was expressed in a high level after 4 h. SDS-PAGE and Western blotting showed a specific band of about 29 kDa (Fig. 2a). The recombinant protein was purified using Ni-NTA affinity columns, and a single band of the same molecular weight was demonstrated (Fig. 2b).

Proliferation of peripheral blood lymphocytes The cell-mediated proliferation patterns in response to Con A and profilin are shown in Fig. 3a and b. The lymphocyte proliferation response was significantly higher in all vaccinated groups than in the control group (P<0.05). Both the medium (50 μg, group 4) and high (100 μg, group 5) dosages of profilin with 50 μg ginsomes could

Statistical analysis All data were analyzed using the software SPSS 15.0 for Windows. The antibody levels, lymphocyte responses measured by MTT, IL-1 production, BWG, and lesion score were expressed as the mean±SD. The mean values were compared by one-way ANOVA analysis of variance followed by the Duncan’s multiple range tests. The difference was considered significant between two groups if P<0.05.

Results Expression and purification of protein The E. coli BL21 (DE3) was transformed with the recombinant vector pET-30a-profilin and was induced by isopropyl-

Fig. 2 SDS-PAGE (a), Western blot of the profilin gene expression in E. coli (b), and identification of the 6× His-profilin fusion protein by Ni-NTA reagent through purifying process (c). a M Protein molecular weight marker; lane 1 pET-30a-profilin BL21 control; lanes 2–5 pET30a-profilin BL21 induced for 2, 4, 6, and 8 h, respectively; lanes 6 pET-30a BL21 control; lane 7 pET-30a BL21 induced for 4 h. b Western blotting of pET-30a-profilin BL21 induced for 6 h. c M protein molecular weight marker; lane 1 the solvent production of the aim protein absorbed by Ni-NTA reagent

Parasitol Res (2012) 110:2445–2453

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only was measured significantly higher than that in group injected with saline on the 14th day post-boost (P < 0.05). However, there was no difference between group immunized with 10 μg profilin plus ginsomes and group immunized with saline. On the 28th day post-booster, the IL-1 production was significantly higher in groups immunized with profilin than that in group immunized with saline (P < 0.05).

Serum antibody responses The results of ELISA performed on serum samples from birds immunized with E. tenella recombinant profilin antigen and control chickens are shown in Fig. 5. Compared with the control group (group 1), a significant increase in mean absorbance values were noted from seventh day post-boost and were still kept at significantly high levels on 42nd day after the birds were boosted with recombinant antigen. Among birds immunized with antigen with or without addition of ginsomes (groups 2–

Fig. 3 Mean (SD) OD stimulated with Con A (a) or profilin fusion protein (b) lymphocyte cultures measured by MTT (n05). Five groups of non-immune chickens were vaccinated subcutaneously with saline control, recombinant profilin antigen (100 μg), and recombinant profilin antigen (10, 50, and 100 μg) mixed with 50 μg ginsomes per dose, respectively. The cells were labeled for 4 h with MTT after 56 h incubation and then mixed with 150 μl dimethyl sulfoxide (DMSO) per well just before measurement. Results were detected at 570 nm using an ELISA reader. Values with different letters differ within time point at P<0.05

significantly enhance the proliferative response to stimulation of recombinant profilin, as well as Con A, when compared with group 2 immunized with 100 μg recombinant profilin alone. IL-1 production Cytokine secretion of lymphocytes is shown in Fig. 4. IL-1 production in both groups immunized with profilin plus ginsomes and the group immunized profilin

Fig. 4 Inductive activity of interleukine-1 (IL-1) with the vaccine. Five groups of non-immune chickens were vaccinated subcutaneously with saline control, recombinant profilin antigen (100 μg), and recombinant profilin antigen (10, 50, and 100 μg) mixed with 50 μg ginsomes per dose, respectively. Results are expressed as means of the OD570 ±SD. Values with different letters differ within time point at P< 0.05

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plus ginsomes) was significantly lower than that of group C (100 μg profilin only) (P < 0.05). There were 32.61%, 37.68%, and 42.27% protection rate in chickens immunized with profilin plus ginsomes (groups D– F), respectively. The chickens immunized with antigen alone (group C) had 37.20% protection rate (Fig. 6).

Discussion

Fig. 5 ELISA analysis of the sera antibody responses of chickens immunized with recombinant profilin antigen: group 1, saline control; group 2, chickens were vaccinated subcutaneously with 100 μg recombinant profilin antigen; groups 3–5 were vaccinated subcutaneously with 50 μg ginsomes mixed 10, 50, and 100 μg recombinant profilin antigen per dose, respectively. Values with different letters differ within time point at P<0.05

5), a significantly lower level in mean absorbance values in group 2 (only high dose of antigen but without ginsomes) was noted (P<0.05). Immune protective efficacy To determine the total efficacy of a vaccine candidate on coccidiosis, the rate of survival, BWG, oocysts per gram (OPGs), and lesion scores after homologous oocysts challenge were measured. Rate of survival of all these groups was 100%. The BWG of the birds in seven groups are presented in Table 1. At seventh day after challenge with E. tenella, BWG in group A was significantly lower than that in other groups, and BWG in group C was significantly lower than that in groups D–G (P<0.05). There were no significant difference of BWG among groups B, D, E, F, and G (P>0.05). The cecum lesions at seventh day after challenge with E. tenella were estimated (see Table 2). The mean lesion scores in groups D–F were 0.4, 0.3, and 0.3, respectively. The chickens immunized with antigen alone (group C) had mean lesion scores of 0.5. The unimmunized controls (group A) had a mean lesion score of 0.7. The lesion score in group A was significantly higher than in other groups (P<0.05). The lesion score in groups B, D, E, and F were significantly lower than that of groups A and C (P<0.05). No significant difference was observed in lesion scores among group B, D, E, and F (P>0.05). The cecum content of each chicken was collected to analyses OPG and protection rate of each group. As shown in Fig. 6, the OPG of group F (100 μg profilin

More and more E. tenella antigens are found to be efficient as vaccine candidates with or without specific adjuvant (Song et al. 2009c; Subramanian et al. 2008; Xu et al. 2008; Li et al. 2011; Ding et al. 2008). Profilin is expressed in all sexual stages of E. tenella, stimulates cell-mediated immunity, and contains a putative conserved domain for the actin-regulatory protein profilin (Ding et al. 2004; Jang et al. 2010; Lee et al. 2010b; Song et al. 2000; Lillehoj et al. 2000; Zhao et al. 2011). In the present study, we investigated the adjuvant effect of ginsomes on the immune response and protection efficacy induced by recombinant profilin antigen plus ginsomes after oral infection with E. tenella sporulated oocysts. There were considerably less lesions in experimental chickens in the subunit vaccine plus ginsomes. In our previous trials, 50 μg ginsomes enhanced the immune response better than other dosages and were thus chosen for this study. A significantly higher BWG indicated that vaccination with or without the adjuvant provided protection against challenge. Ten micrograms of profilin plus ginsomes was more efficient than 100 μg profilin alone, which suggests that ginsomes enhance the immune protection. Although vaccinated groups had lower BWG than the medicated group, the difference was not significantly. Cell-mediated immunity plays a key role in the protection against E. tenella in chickens, but the role of humoral immunity in E. tenella infected chickens is ambiguous (Jang et al. 2010; Ding et al. 2004; Wallach 2010; Abdul Hafeez et al. 2006). Chickens in the subunit vaccine plus ginsomes groups had higher antibody titers than those of the subunit vaccine group before challenge. In this study, the chickens immunized by vaccine plus ginsomes were found to be significantly enhanced in lymphocyte proliferation induced by Con A. This finding indicates that peripheral blood lymphocyte activity was maintained after the challenge, and that the cell-mediated immunity involving T cells seems to contribute to the protection against E. tenella. Cytokines mediate and modulate host immune responses to infectious disease and activate either

Parasitol Res (2012) 110:2445–2453 Table 2 Cecum lesions examined at 8 days after challenge with E. tenella

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Groups

A B

Results are expressed as the number of chicks in different intestinal lesion ranks. Values with different superscripts differ significantly (P<0.05)

C D E F G

Treatment

Intestinal lesion ranks

Unimmunized challenged unmedicated control Unimmunized challenged and diclazuril medicated 100 μg protein 10 μg protein+50 μg ginsomes 50 μg protein+50 μg ginsomes 100 μg protein+50 μg ginsomes Unimmunized unchallenged unmedicated control

humoral or cell-mediated immunity, depending on the different cytokines induced (Lowenthal et al. 2000). Previous studies demonstrated that E. tenella induced a CD4+ T cell, macrophage response, and an increase in the IL-2, IL-4, IL-8, IL-10, IL-18, and INF-γ response in the cecum (Cornelissen et al. 2009). Vaccinating chickens or embryo with pcDNA-profilin vaccine plus IL-1 or IL-1β can reduce fecal oocyst shedding and enhance serum IgG antibody level and humoral response (Lillehoj et al. 2005; Min et al. 2002; Ding et al. 2004). Lee et al. (2010a) observed an enhanced transcriptional level of IL-1β when injecting profilin with QCDC than profilin or QCDC only. In this study, vaccinating with profilin protein plus adjuvant increased IL-1; however,

Fig. 6 The OPG (mean±SD) and protection rate following E. tenella infection. group A, unimmunized challenged unmedicated control; group B, unimmunized challenged medicated with diclazuril; group C, 100 μg antigen; group D, 10 μg antigen+50 μg ginsomes; group E, 50 μg antigen+50 μg ginsomes; group F, 100 μg antigen+50 μg ginsomes; group G, unimmunized unchallenged unmedicated control

Lesion scores (means±SD)

0

1

2

3

4

14

11

4

1

0

0.7±0.83a

23

7

0

0

0

0.2±0.43cd

18 20 22 22 30

10 9 7 8 0

2 1 1 0 0

0 0 0 0 0

0 0 0 0 0

0.5±0.63b 0.4±0.56bc 0.3±0.53bc 0.3±0.45bc 0.0±0.00d

the role of IL-1 in the immunity to Eimeria infection is not clear. Recently plant extracts were proposed as vaccine adjuvants (Berezin et al. 2010; Ogbe et al. 2009). Ginseng, the root of P. ginseng, has been used as a traditional medicine in oriental countries for thousands of years. Previous investigations have found that supplement of Ginseng saponins adjuvant in a commercial vaccine or subunit vaccine candidate can significantly enhance immune responses (Du et al. 2005). Song and Hu (2009) reported the synergistic effect of saponins extracted from ginseng and oil emulsion on immune responses. When saponins were coadministered with oil emulsion, inactive foot-and-mouth disease virus antigen can induce significantly higher antibody response, cytokine production, and lymphocyte proliferation. Similar synergistic effect were also reported by Rivera et al. (2003) when ginseng and aluminum hydroxide mixture were injected as adjuvants. Our studies showed that ginsomes stimulate the immune response. Indeed, not only higher antibody titers but also a higher lymphocyte proliferation response were observed and were coincident with reduced lesion scores. The results of this study indicated that vaccination with profilin plus ginsomes induced an antigen-specific antibody response, induced changes in the local lymphocyte subpopulations, and significantly reduced the total oocysts production following challenge infection. E. tenella is one of the most pathogenic coccidian parasite species, but there are several other species that are also harmful to chicken and highly conserved antigen may induce cross-protection between different species (Belli et al. 2009). Further studies are needed to optimize profilin vaccines and to elaborate whether protection against challenge with other species of Eimeria can be achieved. Although ginsomes appear to be suited as adjuvant for subunit vaccines, further research is necessary to understand the mode of action of ginsomes.

2452 Acknowledgments This work was supported by “the Fundamental Research Funds for the Central Universities” and by a grant from the Science and Technology Department of Jiaxing, Zhejiang Province, China (no. 2007BY6007). The authors thank the team of parasite laboratory, College of Animal Sciences, Zhejiang University.

References Abdul Hafeez M, Akhtar M, Hussain I (2006) Protective effect of eggpropagated Eimeria tenella (local isolates) gametocytes as vaccine(s) against mixed species of coccidia in chickens. Parasitol Res 98(6):539–544 Allen PC, Fetterer RH (2002) Recent advances in biology and immunobiology of Eimeria species and in diagnosis and control of infection with these coccidian parasites of poultry. Clin Microbiol Rev 15(1):58–65 Belli SI, Ferguson DJP, Katrib M, Slapetova I, Mai K, Slapeta J, Flowers SA, Miska KB, Tomley FM, Shirley MW, Wallach MG, Smith NC (2009) Conservation of proteins involved in oocyst wall formation in Eimeria maxima, Eimeria tenella and Eimeria acervulina. Int J Parasitol 39(10):1063–1070 Berezin VE, Bogoyavlenskyi AP, Khudiakova SS, Alexuk PG, Omirtaeva ES, Zaitceva IA, Tustikbaeva GB, Barfield RC, Fetterer RH (2010) Immunostimulatory complexes containing Eimeria tenella antigens and low toxicity plant saponins induce antibody response and provide protection from challenge in broiler chickens. Vet Parasitol 167 (1):28–35 Chapman HD, Cherry TE, Danforth HD, Richards G, Shirley MW, Williams RB (2002) Sustainable coccidiosis control in poultry production: the role of live vaccines. Int J Parasitol 32(5):617–629 Chiani P, Bromuro C, Cassone A, Torosantucci A (2009) Anti-betaglucan antibodies in healthy human subjects. Vaccine 27(4):513–519 Cornelissen JBWJ, Swinkels WJC, Boersma WA, Rebel JMJ (2009) Host response to simultaneous infections with Eimeria acervulina, maxima and tenella: A cumulation of single responses. Vet Parasitol 162(1–2):58–66 Dalloul RA, Lillehoj HS (2006) Poultry coccidiosis: recent advancements in control measures and vaccine development. Expert Rev Vaccines 5(1):143–163 Ding XC, Lillehoj HS, Quiroz MA, Bevensee E, Lillehoj EP (2004) Protective immunity against Eimeria acervulina following in ovo immunization with a recombinant subunit vaccine and cytokine genes. Infect Immun 72(12):6939–6944 Ding J, Bao W, Liu Q, Yu Q, Abdille MH, Wei Z (2008) Immunoprotection of chickens against Eimeria acervulina by recombinant alpha-tubulin protein. Parasitol Res 103(5):1133–1140 Du AF, Hu SH, Wang SH (2005) Eimeria tenella: Ginsenosidesenhanced immune response to the immunization with recombinant 5401 antigen in chickens. Exp Parasitol 111(3):191–197 Fetterer RH, Miska KB, Jenkins MC, Barfield RC (2004) A conserved 19-kDa Eimeria tenella antigen is a profilin-like protein. J Parasitol 90(6):1321–1328 Hu S, Concha C, Lin F, Waller KP (2003) Adjuvant effect of ginseng extracts on the immune responses to immunisation against Staphylococcus aureus in dairy cattle. Vet Immunol Immunop 91 (1):29–37 Jang SI, Lillehoj HS, Lee SH, Lee KW, Park MS, Cha SR, Lillehoj EP, Subramanian BM, Sriraman R, Srinivasan VA (2010) Eimeria maxima recombinant Gam82 gametocyte antigen vaccine protects against coccidiosis and augments humoral and cell-mediated immunity. Vaccine 28(17):2980–2985 Jenkins M, Klopp S, Ritter D, Miska K, Fetterer R (2010) Comparison of Eimeria species distribution and salinomycin resistance in

Parasitol Res (2012) 110:2445–2453 commercial broiler operations utilizing different coccidiosis control strategies. Avian Dis 54(3):1002–1006 Johnson J, Reid WM (1970) Anticoccidial drugs - lesion scoring techniques in battery and floor-pen experiments with chickens. Exp Parasitol 28(1):30–36 Lee SH, Lillehoj HS, Jang SI, Hong YH, Min W, Lillehoj EP, Yancey RJ, Dominowski P (2010a) Embryo vaccination of chickens using a novel adjuvant formulation stimulates protective immunity against Eimeria maxima infection. Vaccine 28(49):7774–7778 Lee SH, Lillehoj HS, Jang SI, Lee KW, Yancey RJ, Dominowski P (2010b) The effects of a novel adjuvant complex/Eimeria profilin vaccine on the intestinal host immune response against live E. acervulina challenge infection. Vaccine 28(39):6498–6504 Li J, Zheng J, Gong P, Zhang X (2011) Efficacy of Eimeria tenella rhomboid-like protein as a subunit vaccine in protective immunity against homologous challenge. Parasitol Res. doi:10.1007/ s00436-011-2603-1 Lillehoj HS (1998) Role of T lymphocytes and cytokines in coccidiosis. Int J Parasitol 28(7):1071–1081 Lillehoj HS, Choi KD, Jenkins MC, Vakharia VN, Song KD, Han JY, Lillehoj EP (2000) A recombinant Eimeria protein inducing interferon-gamma production: comparison of different gene expression systems and immunization strategies for vaccination against coccidiosis. Avian Dis 44(2):379–389 Lillehoj HS, Ding X, Quiroz MA, Bevensee E, Lillehoj EP (2005) Resistance to intestinal coccidiosis following DNA immunization with the cloned 3-1E Eimeria gene plus IL-2, IL-15, and IFNgamma. Avian Dis 49(1):112–117 Lowenthal JW, Lambrecht B, van den Berg TP, Andrew ME, Strom ADG, Bean AGD (2000) Avian cytokines—the natural approach to therapeutics. Dev Comp Immunol 24(2–3):355–365 Ma DX, Ma CL, Pan L, Li GX, Yang JH, Hong JH, Cai HF, Ren XF (2011) Vaccination of chickens with DNA vaccine encoding Eimeria acervulina 3-1E and chicken IL-15 offers protection against homologous challenge. Exp Parasitol 127(1):208–214 McDonald V, Shirley MW (2009) Past and future: vaccination against Eimeria. Parasitology 136(12):1477–1489 Michels MG, Bertolini LCT, Esteves AF, Moreira P, Franca SC (2011) Anticoccidial effects of coumestans from Eclipta alba for sustainable control of Eimeria tenella parasitosis in poultry production. Vet Parasitol 177(1–2):55–60 Min W, Lillehoj HS, Burnside J, Weining KC, Staeheli P, Zhu JJ (2002) Adjuvant effects of IL-1beta, IL-2, IL-8, IL-15, IFNalpha, IFN-gamma TGF-beta4 and lymphotactin on DNA vaccination against Eimeria acervulina. Vaccine 20(1–2):267–274 Ogbe AO, Atawodi SE, Abdu PA, Sannusi A, Itodo AE (2009) Changes in weight gain, faecal oocyst count and packed cell volume of Eimeria tenella-infected broilers treated with a wild mushroom (Ganoderma lucidum) aqueous extract. J S Afr Vet Assoc 80(2):97–102 Qu DF, Yu HJ, Liu Z, Zhang DF, Zhou QJ, Zhang HL, Du AF (2011) Ginsenoside Rg1 enhances immune response induced by recombinant Toxoplasma gondii SAG1 antigen. Vet Parasitol 179(1– 3):28–34 Rivera E, Hu S, Concha C (2003) Ginseng and aluminium hydroxide act synergistically as vaccine adjuvants. Vaccine 21(11–12):1149–1157 Rose ME, Mockett AP (1983) Antibodies to coccidia: detection by the enzyme-linked immunosorbent assay (ELISA). Parasite Immunol 5(5):479–489 Shirley MW, Ivens A, Gruber A, Madeira AMBN, Wan KL, Dear PH, Tomley FM (2004) The Eimeria genome projects: a sequence of events. Trends Parasitol 20(5):199–201 Song X, Hu S (2009) Adjuvant activities of saponins from traditional Chinese medicinal herbs. Vaccine 27(36):4883–4890 Song KD, Lillehoj HS, Choi KD, Yun CH, Parcells MS, Huynh JT, Han JY (2000) A DNA vaccine encoding a conserved Eimeria

Parasitol Res (2012) 110:2445–2453 protein induces protective immunity against live Eimeria acervulina challenge. Vaccine 19(2–3):243–252 Song X, Xu L, Yan R, Huang X, Shah MA, Li X (2009a) The optimal immunization procedure of DNA vaccine pcDNA-TA4-IL-2 of Eimeria tenella and its cross-immunity to Eimeria necatrix and Eimeria acervulina. Vet Parasitol 159(1):30–36 Song X, Zang L, Hu S (2009b) Amplified immune response by ginsenoside-based nanoparticles (ginsomes). Vaccine 27(17):2306– 2311 Song XM, Bao SJ, Wu LH, Hu SH (2009c) Ginseng stem-leaf saponins (GSLS) and mineral oil act synergistically to enhance the immune responses to vaccination against foot-and-mouth disease in mice. Vaccine 27(1):51–55 Subramanian BM, Sriraman R, Rao NH, Raghul J, Thiagarajan D, Srinivasan VA (2008) Cloning, expression and evaluation of the efficacy of a recombinant Eimeria tenella sporozoite antigen in birds. Vaccine 26(27–28):3489–3496 Vermeulen AN, Schaap DC, Schetters TP (2001) Control of coccidiosis in chickens by vaccination. Vet Parasitol 100(1–2):13–20 Wallach M (2010) Role of antibody in immunity and control of chicken coccidiosis. Trends Parasitol 26(8):382–387

2453 Williams RB (1999) A compartmentalised model for the estimation of the cost of coccidiosis to the world's chicken production industry. Int J Parasitol 29(8):1209–1229 Williams RB (2002) Anticoccidial vaccines for broiler chickens: pathways to success. Avian Pathol 31(4):317–353 Xu Q, Song X, Xu L, Yan R, Shah MA, 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(3–4):319– 323 Yan W, Liu X, Shi T, Hao L, Tomley FM, Suo X (2009) Stable transfection of Eimeria tenella: constitutive expression of the YFP-YFP molecule throughout the life cycle. Int J Parasitol 39 (1):109–117 Yang Z, Chen A, Sun H, Ye Y, Fang W (2007) Ginsenoside Rd elicits Th1 and Th2 immune responses to ovalbumin in mice. Vaccine 25 (1):161–169 Zhao Y, Wang C, Lu Y, Amer S, Xu P, Wang J, Lu J, Bao Y, Deng B, He H, Qin J (2011) Prokaryotic expression and identification of 31E gene of merozoite surface antigen of Eimeria acervulina. Parasitol Res 109(5):1361–1365