Journal Pre-proof

Evaluation of the immunoprotective effect of the recombinant Eimeria intestinalis

rhoptry protein 25 and rhoptry protein 30 on New Zealand rabbits

Ge Hao, Changming Xiong, Jie Xiao, Wei He, Yuhua Zhu, Liwen Xu, Qing Jiang,
Guangyou Yang

PII: S0014-4894(24)00022-5
DOI: https://doi.org/10.1016/j.exppara.2024.108719
Reference: YEXPR 108719

To appear in: Experimental Parasitology

Received Date: 9 October 2023

Revised Date: 12 February 2024
Accepted Date: 13 February 2024

Please cite this article as: Hao, G., Xiong, C., Xiao, J., He, W., Zhu, Y., Xu, L., Jiang, Q., Yang, G.,
Evaluation of the immunoprotective effect of the recombinant Eimeria intestinalis rhoptry protein
25 and rhoptry protein 30 on New Zealand rabbits, Experimental Parasitology (2024), doi: https://
doi.org/10.1016/j.exppara.2024.108719.

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© 2024 Published by Elsevier Inc.

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1 Evaluation of the immunoprotective effect of the
2 recombinant Eimeria intestinalis Rhoptry Protein 25
3 and Rhoptry Protein 30 on New Zealand rabbits
a, 1 a, 1
4 Ge Hao , Changming Xiong , Jie Xiao a, Wei He a, Yuhua Zhu a, Liwen Xu a,
5 Qing Jiang b, **, Guangyou Yang a, *
6
a
7 Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural
8 University, Wenjiang, 611130, China

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b
9 Ya'an Polytechnic College, Yaan, 625014, China
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* Corresponding author. Department of Parasitology, College of Veterinary Medicine,
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12 Sichuan Agricultural University, Wenjiang, 611130, China
13 ** Corresponding author. Ya'an Polytechnic College, Yaan, 625014, China
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14 Email addresses: jiangqing0904@126.com (Q. Jiang), guangyou1963@126.com (G.

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15 Yang),
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16 Equal contribution.
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17
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18 Highlights
19 • Recombinant subunit vaccines comprising EiROP25 and EiROP30 were
20 created
21 • Rabbits were immunized with the vaccines and challenged with E. intestinalis
22 • Immunized rabbits had increased weight, anticoccidial indexes, and IgG
23 • Immunized rabbits showed reduced oocyst counts and intestinal lesions
24 • EiROP25 and EiROP30 can generate a moderate level of immune protection

25 Abstract
26 Background: Rabbit coccidiosis is a parasitism caused by either one or multiple
27 co-infections of Eimeria species. Among them, Eimeria intestinalis is the primary
28 pathogen responsible for diarrhea, growth retardation, and potential mortality in
29 rabbits. Concerns regarding drug resistance and drug residues have led to the
30 development of recombinant subunit vaccines targeting Eimeria species as a
31 promising preventive measure. The aim of this study was to assess the
32 immunoprotective efficacy of recombinant subunit vaccines comprising EiROP25 and
33 EiROP30 (rhoptry proteins (ROPs)) against E. intestinalis infection in rabbits.
34 Methods: Cloning, prokaryotic expression, and protein purification were performed
35 to obtain EiROP25 and EiROP30. Five groups of fifty 35-day-old Eimeria-free
36 rabbits were created (unchallenged control group, challenged control group, vector
37 protein control group, rEiROP25 group, and rEiROP30 group), with 10 rabbits in

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38 each group. Rabbits in the rEiROP25 and rEiROP30 groups were immunized with the
39 recombinant proteins (100 μg per rabbit) for primary and booster immunization (100

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μg per rabbit) at a two-week intervals, and challenged with 7 × 104 oocysts per rabbit
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41 after an additional two-week interval. Two weeks after the challenge, the rabbits were
42 euthanized for analysis. Weekly collections of rabbit sera were made to measure
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43 changes in specific IgG and cytokine level. Clinical symptoms and pathological
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44 changes after challenge were observed and recorded. At the conclusion of the animal
45 experiment, lesion scores, the relative weight increase ratio, the oocyst reduction rate,
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46 and the anticoccidial index were computed.

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47 Results: Rabbits immunized with rEiROP25 and rEiROP30 exhibited relative weight
48 gain ratios of 56.57% and 72.36%, respectively. Oocysts decreased by 78.14% and
49 84.06% for the rEiROP25 and rEiROP30 groups, respectively. The anticoccidial
50 indexes were 140 and 155. Furthermore, there was a noticeable drop in intestinal
51 lesions. After the primary immunization with rEiROP25 and rEiROP30, a week later,
52 there was a notable rise in specific IgG levels, which remained elevated for two weeks
53 following challenge (P < 0.05). Interleukin (IL)-2 levels increased markedly in the
54 rEiROP25 group, whereas IL-2, interferon gamma (IFN-γ), and IL-4 levels increased
55 substantially in the rEiROP30 group (P < 0.05).
56 Conclusion: Immunization of rabbits indicated that both rEiROP25 and rEiROP30
57 are capable of inducing an increase in specific antibody levels. rEiROP25 triggered a
58 Th1-type immune protection response, while rEiROP30 elicited a Th1/Th2 mixed
59 response. EiROP25 and EiROP30 can generate a moderate level of immune
60 protection, with better efficacy observed for EiROP30. This study provides valuable
61 insights for the promotion of recombinant subunit vaccines targeting rabbit E.
62 intestinalis infection.
63 Keywords: Rabbit; Eimeria intestinalis; Rhoptry protein 25; Rhoptry protein 30;
64 Recombinant subunit vaccine
65

66

67

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68 1. Introduction
69

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Rabbit coccidiosis is a disease caused by Eimeria. spp. infection of the intestinal
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71 or hepatic epithelial cells of rabbits, with Eimeria intestinalis being one of the most
72 dangerous species (Pakandl, 2009). Coccidiosis leads to serious symptoms, including
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73 diarrhea, stunted growth, and even death, resulting in huge economic damage to the
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74 rabbit farming industry (Li et al., 2019; Tao et al., 2017). Currently, the primary
75 anticoccidial strategy is the addition of anti-coccidial drugs to rabbit feed or drinking
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76 water to mitigate the impact of the disease (Abbas et al., 2011). However, the
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77 prolonged use of these drugs has resulted in considerable drug resistance and drug
78 residues (Flores et al., 2022). To effectively prevent and control rabbit coccidiosis,
79 vaccination is considered one of the most promising approaches. Recent research has
80 focused primarily on attenuated live vaccines and recombinant subunit vaccines
81 (Shirley et al., 2007). Studies have shown that various vaccine types have
82 demonstrated good efficacy in preventing coccidiosis (Chen et al., 2023; Li et al.,
83 2021, 2019; Sadek Bachene et al., 2018; Xiao et al., 2023a). Some phytochemical
84 extracts can also have a positive impact on the control of rabbit coccidiosis(Eladl et
85 al., 2020; Konmy et al., 2023; Metwally et al., 2022). Live vaccines possess strong
86 immunogenicity, but are costly and time-consuming to produce because of the
87 inability to generate oocysts outside the host and they carry a risk of reversion to
88 virulence (Soutter et al., 2020). By contrast, recombinant subunit vaccines, offer
89 simplified production, safety, and stability, making them an attractive alternative.
90 Antigen selection, however, constitutes a pivotal step in developing recombinant
91 subunit vaccines (Peek and Landman, 2011).
92 The apicomplexan parasite possesses a unique apical organelle system consisting
93 of microtubules, rhoptries, and dense granules, among which the rhoptries are
94 rod-shaped, with two distinct regions, the neck and the base (Engelberg et al., 2020).
95 Rhoptry proteins, specifically the rhoptry kinase family proteins, are secretory
96 virulence factors that are crucial for cellular invasion, parasitophorous vacuole
97 formation, and host cell modulation (Dogga et al., 2017; Kemp et al., 2013). Studies

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98 have reported that rhoptry proteins (ROPs) participate considerably in the virulence
99 and pathogenicity of apicomplexan parasites (Wu et al., 2022) and are promising

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vaccine candidates (Mongui et al., 2009; Pastor-Fernández et al., 2015; Song et al.,
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101 2020). However, there is currently insufficient research on the use of ROPs as
102 antigens for recombinant subunit vaccines targeting rabbit E. intestinalis (Xiao et al.,
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103 2023; Xiong et al., 2023).

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104 Herein, we prokaryotically expressed E. intestinalis ROP25 and ROP30, which

105 were selected from the transcriptomic data of E. intestinalis, and conducted
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106 immunoprotection experiments using recombinant subunit vaccines, rEiROP25 and

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107 rEiROP30. This study provides a guide for the further advancement of subunit
108 vaccines for E. intestinalis.
109

110 2. Materials and methods

111

112 2.1. Parasites and animals

113

114 E. intestinalis oocysts were passaged and stored at 4 °C under the supervision of
115 the Department of Parasitology of Sichuan Agricultural University. The department
116 also supplied the four developmental stages of E. intestinalis, encompassing
117 unsporulated oocysts, sporulated oocysts, schizonts, and gametocytes. Negative serum
118 samples (collected from 1-month-old rabbits without Eimeria infection) and positive
119 serum samples (collected from experimental rabbits on the 7th day after challenge) for
120 E. intestinalis were sourced from the same department.
121 Fifty coccidian-free New Zealand rabbits (35 days old, weighing 0.874 ± 0.146
122 kg, 50-50 male-female ratio) were provided by the Department of Parasitology of
123 Sichuan Agricultural University. References to methods of breeding New Zealand
124 rabbits(Wei et al., 2020). The rabbit cages, feeding utensils, and other equipment were
125 sterilized by baking, and the feed was subjected to high-temperature drying. The
126 rabbits had unlimited access to food and water, and the water was infused with

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127 Diclazuril. A week before challenge, the use of anti-coccidial drugs was discontinued,
128 and daily parasitological examinations were performed to ensure the absence of

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131 2.2. Cloning and bioinformatic analysis of EiROP25 and EiROP30

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132
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133 EiROP25 and EiROP30 were screened using BLAST in combination with
134 transcriptome data. Open reading frames (ORFs) and amino acid sequences were
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135 obtained using ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/). Total RNA of

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136 E. intestinalis (non-sporulating oocysts, sporulating oocysts, cleistogamy, and

137 gametophytes) was extracted using a commercial kit (Tiangen, Beijing, China) and
138 cDNA was synthesized (Thermo Fisher Scientific, Waltham, MA, USA). The
139 resulting cDNA was then mixed and used as a template for PCR amplification.
140 EiROP25 and EiROP30 specific forward (F) and reverse (R) primers were designed
141 based on the E. intestinalis transcriptome data: EiROP25: F5'-
142 CGGGATCCATGAAGCCTTTGAGGCTGATGG-3' and EiROP25: R5'-
143 CCCTCGAGTTAGCACGTCATCCCCACCG-3'; EiROP30-F5'-
144 CGGGATCCATGCCCAAAATGTGCTCAGACG-3' and EiROP30-R5'-
145 CCCTCGAGTTAGCCGATCTCCCATTTAAGG-3' and contained BamHI and XhoI
146 restriction endonuclease sites (Takara, Dalian, China).
147 The molecular weights of the predicted proteins were determined using the
148 ExPASy proteomics server. Transmembrane regions and signal peptides were
149 identified using the TMHMM Server v.2.0 and SignalP 4.1 server, respectively. B-cell
150 epitopes were predicted using the Immune Epitope Database Analysis Resource.
151 Additionally, multiple sequence comparisons were carried out using Jalview 2.11.2.0
152 (Xiao et al., 2023a). Antigen toxicity was predicted using
153 (https://webs.iiitd.edu.in/raghava/toxinpred2/batch.html), and antigen sensitization
154 was predicted using (https://webs.iiitd.edu.in/raghava/algpred2/batch.html).
155

156 2.3. Expression and purification

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158 Using double digestion, the expression constructs pMD19-T-EiROP25 and

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pMD19-T-EiROP30 plasmids were ligated into the pET-32a (+) vector (Tiangen). The
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160 constructed pET-32a (+) - EiROP25 and pET-32a (+) - EiROP30 were transformed
161 into Escherichia coli BL21 (DE3) and stimulated using Isopropyl
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162 β-d-1-thiogalactopyranoside (IPTG) (Sigma, Cream Ridge, NJ, USA) at 37 °C for 8 h.

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163 To confirm the expression of the recombinant proteins, bacterial inclusion bodies
164 were collected, and the supernatants and precipitates were examined using sodium
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165 dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An Ni2+ affinity

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166 chromatography column (Qiagen, Hilden, Germany) was used to purify the
167 recombinant proteins, and 12% SDS-PAGE (Solarbio, Beijing, China) was used to
168 verify their purity.
169

170 2.4. Western blotting

171

172 The purified and separated rEiROP25 and rEiROP30 were transferred onto
173 polyvinylidene fluoride (PVDF) membranes (Boster, Wuhan, China). For
174 immunoblotting analysis of recombinant proteins, E. intestinal positive/negative
175 rabbit serum (diluted at 1:100) was utilized as the primary antibody, and horseradish
176 peroxidase (HRP)-labeled goat anti-rabbit IgG (diluted at 1:1000) served as the
177 secondary antibody.
178

179 2.5. Immunization and challenge

180

181 Fifty rabbits aged 35 days without coccidia infection were randomly divided into
182 five groups of 10 rabbits each: The challenged control group, the unchallenged
183 control group, the vector protein control group, the rEiROP25 group, and the
184 rEiROP30 group (Table 1). The vector protein control group was immunized with
185 pET-32a empty protein (100 g/each), the immunized recombinant protein group was
186 immunized with rEiROP25 or rEiROP30 (100 g/each), and the other groups were

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187 injected with 1 ml of phosphate-buffered saline (PBS). After the initial immunization,
188 a second immunization was administered two weeks later. Subsequently, two weeks

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after the second immunization, all groups except for the unchallenged control group
were exposed to E. intestinalis challenge (7 × 104 oocytes per rabbit). The rabbits
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191 were sacrificed and sampled two weeks after the challenge (Figure 1).
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192
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193 2.6. Evaluation of the protective effects

194
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195 The rabbits were weighed individually before the first immunization, before the
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196 second immunization, and before dissection, to calculate the average weight gain:
197 ΔW1 (weight at challenge - weight before the first immunization), ΔW2 (weight
198 before execution - weight at challenge), and the relative weight gain rate (average
199 ΔW2 of the experimental group/average ΔW2 of the unchallenged control group ×
200 100%). After challenge, the mortality rate of each group was recorded, and the clinical
201 symptoms, such as poor mental health, loss of appetite, diarrhea, and abdominal
202 distension, were observed and recorded in the challenged control group and the
203 unchallenged control group.
204 When all the rabbits were sacrificed, 2 g feces were collected from the rectum.
205 The average number of oocysts per gram (OPG) of feces in each group was counted
206 and the oocyst reduction rate was calculated. Oocyst reduction rate = (OPG of the
207 challenged control group - OPG of the immunized group)/OPG of the challenged
208 control group × 100% (Bortoluzzi et al., 2018).
209 All rabbits were sacrificed on day 14 after the 2nd challenge and the posterior
210 segment of the jejunum and the entire ileum were collected. Lesions were scored on
211 five levels from 0 to 4.
212 The anticoccidial index (ACI) is an objective indicator commonly used
213 internationally to evaluate the effectiveness of anticoccidial drugs or vaccines
214 (McManus et al., 1968). ACI = (relative rate of weight gain + survival rate) − (lesion
215 value + oocyst value).
216

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217 2.7. Detection of serum IgG levels
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To observe the changes in specific serum IgG levels in the rabbits after
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220 immunization with the two recombinant proteins, serum was collected from the
221 rabbits at a fixed time every week from before the first immunization until after
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222 challenge until sacrifice. All serum samples were stored at −20 ℃. The serum IgG
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223 levels of the rabbits were measured using an indirect enzyme-linked immunosorbent
224 assay (ELISA) based on rEiROP25 and rEiROP30, respectively( Liu et al., 2018).
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226 2.8. Detection of serum cytokine levels

227

228 Rabbit serum interleukin (IL)-2, IL-4, IL-10, and interferon gamma (IFN-γ)
229 levels were measured using a commercial ELISA kit (Solarbio, Beijing, China)
230 according to the supplier's instructions.
231

232 2.9. Data Analysis

233

234 The data are presented as the mean ± standard deviation (SD), and group
235 differences were assessed using one-way analysis of variance (ANOVA) conducted
236 using IBM SPSS Statistics 22.0 (IBM Corp., Armonk, NY, USA. A significance level
237 of P < 0.05 was considered statistically significant, while P < 0.01 was deemed highly
238 significant. GraphPad Prism version 5.0 (GraphPad Software Inc. La Jolla, CA, USA)
239 was employed for data visualization and graph creation.
240

241 3. Results
242

243 3.1. Cloning and Bioinformatics Analysis of EiROP25 and EiROP30

244

245 EiROP25 (GenBank: OQ411012) and EiROP30 (GenBank: OQ411013) were

246 amplified using the mixed cDNAs of the four stages of E. intestinalis as templates,

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247 and the amplified fragment were 528 bp and 387 bp, respectively. Through cloning
248 and sequencing analysis, the amplified gene fragment showed 100% sequence

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similarity to the sequences present in the transcriptome data of E. intestinalis.
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250 The open reading frame of the EiROP25 gene is 528 bp and encodes a putative
251 protein of 176 amino acids. The predicted relative molecular weight is 18.79 kDa and
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252 the calculated isoelectric point (PI) is 6.88. The EiROP30 gene has an open reading
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253 frame of 387 bp and encodes a putative protein of 129 amino acids. The predicted
254 relative molecular weight was 14.58 kDa and the calculated isoelectric point (PI) was
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255 8.90. No signal peptide or transmembrane region was predicted in either protein. The
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256 amino acid sequences of EiROP25 and EiROP30 were compared with other
257 protozoan amino acid sequences in a multiple alignment (Figure 2). As can be seen in
258 the figure, the purple color indicates conserved, and the species name and percentage
259 of amino acid sequence similarity are marked at the beginning. The above two
260 proteins are less conserved in the protozoa. The secondary structure and B-cell
261 antigen epitopes of the two proteins were also predicted and labeled in the figure, with
262 the red and green lines showing the predicted secondary structure and the red dashed
263 boxes showing the B-cell antigen epitopes. Both EiROP25 and EiROP30 were
264 predicted to be free of antigenic toxicity and antigenic sensitization.
265

266 3.2. Expression and purification

267 The successfully constructed pET32a(+)-EiROP25 and pET32a(+)-EiROP30
268 were transformed into E.coli BL21(DE3) for induction of expression at 25 °C, 1 mM
269 IPTG, 8 h. 12% SDS-PAGE gel electrophoresis showed that the expressed rEiROP25
270 protein was about 37 kDa and the rEiROP30 protein was about 35 kDa, both of
271 which were expressed in inclusion bodies (Figure 3).
272

273 3.3. Western Blotting

274

275 Immunoblotting analysis revealed that sera from rabbits infected with E.
276 intestinalis recognized rEiROP25 and rEiROP30 and showed single bands around 37

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277 kDa and 33 kDa, respectively, while no bands were observed using rabbit E.
278 intestinalis negative sera, indicating that both recombinant proteins rEiROP25 and

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rEiROP30 are immunoreactive (Figure 3, lanes 2 and 3).
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281 3.4. Evaluation of protective effects

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282
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283 The challenged control group exhibited a loss of appetite and weight loss
284 approximately one week after challenge. Seven rabbits experienced diarrhea, and
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285 most of them had soft, unformed feces. In contrast, the rEiROP25 and rEiROP30
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286 groups did not display any noticeable clinical signs, with only a few rabbits having
287 soft, unformed feces. Upon dissection, the challenged control group showed
288 significant lesions in the ileum and lower jejunum, while the recombinant protein
289 immunized group had few or no lesions, resulting in significantly reduced lesion
290 scores (P < 0.05) (Figure 4). The relative body weight gain was 56.57% and 72.36%
291 in the rEiROP25 and rEiROP30 groups, respectively, and the reduction in oocysts was
292 78.14% and 84.06%, respectively, with ACI values of 140 and 155, compared with the
293 unchallenged control group (Table 2).
294

295 3.5. Detection of specific antibody levels

296

297 The mean serum IgG antibody levels in the rEiROP25 and rEiROP30 immunized
298 groups increased significantly after the second immunization, peaked one week
299 thereafter, and remained stable (Figure 5). These elevated levels were sustained for up

300 to two weeks after the challenge. The specific IgG antibody level in the challenge
301 control group did not exhibit significant alterations before and after the challenge,
302 staying consistently low. In contrast, the vector protein control group showed a
303 temporary increase followed by a decrease after immunization, with an overall lower
304 value compared to the recombinant protein immunized group. This difference was
305 statistically significant when compared with that of the rEiROP25 and rEiROP30
306 group (P < 0.05).

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308 3.6. Detection of cytokine levels

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310 The IL-2 levels in the group immunized with recombinant protein ROP25 showed a
311 significant difference (P < 0.05) compared with that in the challenged control group,
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312 but no significant differences were observed for the levels of IL-4, IL-10, and IFN-γ
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313 in this comparison. Similarly, significant differences (P < 0.05) were found in the
314 levels of IL-2, IL-4, and IFN-γ between the group immunized with recombinant
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315 protein ROP30 and the challenged control group, with no significant variations noted
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316 for IL-10 levels (Figure 6).

317

318 4. Discussion
319 Coccidiosis poses a threat to the global animal welfare. Recombinant vaccines,
320 overcoming limitations of traditional live vaccines, show promise in controlling
321 Eimeria parasites economically. By utilizing defined antigens and safe adjuvants,
322 these vaccines induce effective immune responses(Venkatas et al., 2019).In Eimeria,
323 ROP proteins are typically detected after sporozoite release and play crucial roles in
324 invasion, modulation of the parasitophorous vacuole, host cell membrane remodeling,
325 and protection against host defenses (Lal et al., 2009). In T. gondii, rhoptry proteins
326 have been extensively investigated as candidate vaccine antigens. Immunizing mice
327 with recombinant viral vectors expressing T. gondii rhoptry 18 induced a notable rise
328 in the specific antibody response (Yoon et al., 2022). Other members of the ROP
329 family have also shown good immune protective efficacy against Toxoplasma
330 infection (Bradley and Sibley, 2007; Dlugonska, 2008; Dongchao et al., 2020; El Hajj
331 et al., 2006; Foroutan et al., 2019). It has been reported that ROP30 of Eimeria tenella,
332 an avian Eimeria species, is a secretory protein during the sporozoite stage and
333 exhibits significantly higher expression levels during the schizont stage, indicating its
334 crucial role in parasite re-invasion and development. Therefore, it holds promise as a
335 candidate vaccine antigen against Eimeria infection (Bingxiang et al., 2022).
336 Recombinant E. tenella ROP17 has been shown to significantly reduce cecal lesions

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337 and oocyst output in chickens and displayed good anti-coccidial efficacy in rabbits in
338 immunization experiments (Xiao et al., 2023a). ROP35, when used as a subunit

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vaccine in immunized chickens, induced high levels of serum IgY, increased body
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340 weight, lowered the cecal lesion scores, and reduced oocyst output compared with
341 those in the challenged control group (Wang et al., 2022). In this study, ROP25 and
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342 ROP30 were used for the first time as antigens in recombinant subunit vaccines.
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343 Animal experiments demonstrated that compared with the challenged control group,
344 rabbits immunized with rEiROP25 and rEiROP30 showed increased average body
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345 weight and decreased oocyst production. The relative weight gains of the rEiROP25
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346 and rEiROP30 groups were 56.57% and 72.36%, respectively, while the oocyst
347 reduction rates were 82.21% and 84.06%, respectively. Although the number of
348 oocysts is not high during the prepatent period of infection, conducting daily fecal
349 oocyst counts during the prepatent period might provide a better observation of the
350 vaccine's impact on the early invasion. Our results indicated that immunization with
351 rEiROP25 and rEiROP30 conferred protective effects against E. intestinalis infection
352 in rabbits, with rEiROP30 exhibiting a better weight gain performance.
353 IFN-γ and IL-2, as Th1 cytokines, are crucial to combat coccidiosis (Chen et al.,
354 2022). IL-2 stimulates the proliferation and maturation of various immune cells
355 (Ivanova et al., 2019). Th2 cytokines, primarily IL-4, stimulate B cell proliferation
356 and differentiation, promoting antibody production and enhancing humoral immune
357 responses (Hoan et al., 2014). Studies have also indicated an increase in IL-4 levels
358 after immunization (Chen et al., 2021; J. Liu et al., 2018, p. 14; T. Liu et al., 2018). In
359 previous reports, transcription of ROP genes was observed in various life cycle stages
360 of the Eimeria. Different ROPs may share common roles in hijacking signaling
361 pathways and immune responses in infected cells, potentially participating in host cell
362 invasion (Ribeiro et al., 2021). For instance, Eimeria tenella Rhoptry Kinase 2 induces
363 early production of schizonts(Ribeiro et al., 2023). Our results showed that after
364 immunization with rEiROP25, the level of IFN-γ did not increase, while the level of
365 IL-2 increased significantly. Therefore, further research is needed to investigate
366 whether rEiROP25 stimulates Th1-mediated immunity. Immunization with rEiROP30

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367 resulted in heightened generation of both IL-2 and IFN-γ, alongside increased IL-
368 4levels. Therefore, rEiROP30 can elicit both Th1 and Th2 immune responses. IL-10

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regulates inflammatory responses and inhibits cytokine production and cellular
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370 immune responses (Wu et al., 2016). In this study, neither rEiROP25 nor rEiROP30
371 caused an increase in IL-10 levels compared with those in the unchallenged control
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372 group. This suggests their potential to enhance IFN-γ-mediated responses, leading to
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373 effective immune clearance of replicating parasites and a subsequent reduction in

374 oocyst shedding, as previously hypothesized (Rothwell et al., 2004).
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375
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376 5. Conclusions
377

378 Immunization of rabbits with rEiROP25 and rEiROP30 provided immune

379 protection against E. intestinalis infection. The relative weight gain of the rEiROP25
380 and rEiROP30 immunized groups was 56.57% and 72.36%, respectively, and the
381 oocyst output was reduced by 78.14% and 84.06%, respectively. rEiROP30
382 demonstrated superior results in relation to weight increase. rEiROP25 and rEiROP30
383 are capable of eliciting both cell-mediated and humoral immune responses, making
384 them promising selections for an anti-E. intestinalis vaccine in rabbits.
385

386 Ethics Approval and Consent to Participate

387 The Animal Care and Use Committee of Sichuan Agricultural University reviewed
388 and approved the animal study protocol (No. SYXK 2019-189). All animal
389 procedures adhered to the Guide for the Care and Use of Laboratory Animals
390 (National Research Council, Bethesda, MD, USA) and the Animal Research:
391 Reporting of In Vivo (ARRIVE) Experiments guidelines
392 (http://www.nc3rs.org.uk/arrive-guidelines). All applicable institutional and national
393 guidelines for the care and use of animals were followed.
394

395 Consent for publication

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396 Not applicable.
397

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398 Funding -p
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399 Supported by the Yaan Science and Technology Program（23ZDYF0004）
400
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401 Contributions
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402 Experimental design: YGY and HG. Experiments: HG, XCM, XJ, HW, ZYH, and
403 XLW. Data analysis: HG. Writing, original draft preparation: HG and XCM. Writing,
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404 review and editing: HG, XCM, JQ, and YGY. All authors read and approved the final
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405 version of the manuscript.

406

407 Declaration of competing interest

408 The authors all certify that they have no competing interests.
409

410 Data availability

411 All data related to this research are present in the manuscripts.
412

413 Acknowledgements
414 The authors thank Luyang Xu, Xiaowei Zhu, Maochuan Guo and Yanxin Chen for
415 their contributions to the animal experiments and sample collection.
416
417 The list of abbreviations
418 Eimeria intestinalis E. intestinalis
419 Rhoptry protein ROP
420 ORFs Open reading frames
421 PVDF polyvinylidene fluoride
422 HRP horseradish peroxidase
423 ELISA enzyme-linked immunosorbent assay
424 ACI The anticoccidial index
425 RONs rhoptry neck proteins

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426

427

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428 -p
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429

430
lP

431
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432 References
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582

583
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585

586

587

588

589 Figure legends

590

591 Figure 1 Immunization, challenging, and sampling procedure

592

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593 Figure 2 Amino acid sequence comparison between EiROP25 and EiROP30 with
594 other protozoan ROP25 and ROP30 proteins.

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(A) Eimeria tenella (UniProt: XP_013228137.1); Eimeria necatrix (UniProt:
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596 XP_013432729.1); Eimeria mitis (UniProt: XP_013355441.1); Eimeria
597 acervuline (UniProt: XP_013248738.1); Eimeria brunetti (UniProt: CDJ51611.1);
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598 (B) Eimeria tenella (UniProt: XP_013228138.1); Eimeria brunetti (UniProt:

na

599 CDJ51611.1); Eimeria necatrix (UniProt: XP_013432728.1); Cyclospora

600 cayetanensis (UniProt: OEH77029.1); Eimeria acervuline (UniProt:
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601 XP_013248738.1). The purple color indicates conserved, and the species name and
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602 percentage of amino acid sequence similarity are marked at the beginning. Red and
603 green lines show the predicted secondary structures and the red dashed boxes show
604 the B-cell antigen epitopes. EiROP25, Eimeria intestinalis rhoptry protein 25;
605 EiROP30, Eimeria intestinalis rhoptry protein 30.
606

607 Figure 3 SDS-PAGE and Western blotting analysis of rEiROP25(A) and rEiROP30
608 (B)
609 Lane: M: protein molecular weight markers (in kDa); lane 1: purified recombinant
610 proteins; lane 2: purified recombinant proteins incubated with anti-E. intestinalis
611 positive serum; lane 3 purified recombinant proteins incubated with negative serum
612 from coccidia-free rabbits. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel
613 electrophoresis; rEiROP25, recombinant Eimeria intestinalis rhoptry protein 25;
614 rEiROP30, recombinant Eimeria intestinalis rhoptry protein 30.
615

616 Figure 4 Gross postmortem examination.

617 (A) Unchallenged control group; (B) Challenged control group: Severe
618 gastrointestinal bleeding was observed; (C) Vector protein group: The presence of
619 intestinal thickening was apparent.; (D) rEiROP25 group: The intestinal wall has
620 slightly thinned.; (E) rEiROP30 group: The intestines are normal. rEiROP25,
621 recombinant Eimeria intestinalis rhoptry protein 25; rEiROP30, recombinant Eimeria
622 intestinalis rhoptry protein 30.

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623

624

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625 -p
Figure 5 The changes in serum anti-rEiROP25 and anti-rEiROP30 levels. rEiROP25,
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626 recombinant Eimeria intestinalis rhoptry protein 25; rEiROP30, recombinant Eimeria
627 intestinalis rhoptry protein 30; OD, optical density; PBS, phosphate-buffered saline,
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628 32a, vector control.

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629

630
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631 Figure 6 Cytokines (IL-2, IL-4, IL-10, and IFN-γ) were measured in serum before
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632 coccidia challenge. Different superscripts (a, b) indicate a significant difference (P <
633 0.05). The same superscript indicates no significant difference (P > 0.05). The
634 concentration unit of cytokines was pg/ml. IL-2, Interleukin 2; IL-4, Interleukin 4;
635 IL-10, Interleukin 10; IFN-γ, interferon gamma; PBS, phosphate-buffered saline, 32a,
636 vector control; ROP25, rhoptry protein 25; ROP30, rhoptry protein 30.
637

638

639

640

641

642

643
644

645

646

647

648

649

650

651 Tables
652 Table 1. Immune procedures.

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Immunization procedures
Rabbit/

r
Group challenge
n -p Immunization
dosage route
ages (days old)
re
Neck
lP

Unchallenged 1 ml
10 subcutaneous 35, 49 -
control PBS
injection
na

7×104
Neck sporulated
Challenged 1 ml
10 subcutaneous 35, 49 oocysts/ 63
ur

control PBS
injection days
old/oral
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100 g
pET- 7×104
32a(+) Neck sporulated
Vector
10 +1mg subcutaneous 35, 49 oocysts/ 63
protein
Quil-A injection days
in 1ml old/oral
PBS
100 g
7×104
rEiROP2
Neck sporulated
5+1mg
rEiROP25 10 subcutaneous 35, 49 oocysts/ 63
Quil-A
injection days
in 1 ml
old/oral
PBS
100 g
7×104
rEiROP3
Neck sporulated
0+1mg
rEiROP30 10 subcutaneous 35, 49 oocysts/ 63
Quil-A
injection days
in 1 ml
old/oral
PBS
653

654

655

656

657

658

659

660 Table 2. Evaluation of the protective effect of proteins against Eimeria intestinalis

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Relative Oocyst Survi
Average Oocyst Mean
weight reducti val
Group weight gain output ( lesion ACI

r
gain ratio on ratio rate
(kg)
(%)
-p 105)
(%)
scores
(%)
re
lP

Unchalleng
0.38 ± 0.02a 100.00% 0a - 0a 100 200
ed control
na

Challenged
0.11 ± 0.04b 28.94% 2.43 ± 0.31b 0 2.3 ± 0.33b 100 66
control
ur

Vector
0.17 ± 0.04b 1.71 ± 0.17b 2 ± 0.36b
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protein 44.73% 29.55% 100 104

control

rEiROP25 0.215 ± 0.09c 56.57% 0.53 ± 0.18c 78.14% 1.5 ± 0.56c 100 140

rEiROP30 0.275 ± 0.07d 72.36% 0.39 ± 0.14d 84.06% 1.6 ± 0.62c 100 155

661 The data are reported as the mean ± standard deviation. Different superscript letters (a,
662 b, c, d; ANOVA, P < 0.05) within each column indicate significant differences
663 between the data, while data marked with the same superscripted letter are not
664 significantly different (P > 0.05).
665
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Declaration of competing interest
The authors all certify that they have no competing interests.

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