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All issues Volume 32 (2025) Parasite, 32 (2025) 76 Full HTML
Open Access
Issue
Parasite
Volume 32, 2025
Article Number 76
Number of page(s) 8
DOI https://doi.org/10.1051/parasite/2025071
Published online 02 December 2025

© W. Chen et al., published by EDP Sciences, 2025

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abbreviations

2-DE: Two-dimensional gel electrophoresis;

ABC: ATP-binding cassette;

PBS: Phosphate-buffered Saline;

PBST: PBS with Tween-20;

IEF: Isoelectric focusing;

IgY: Immunoglobulin of yolk;

IPG: immobilized pH gradient;

MALDI-TOF-MS/MS: Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry/mass spectrometry;

MS: Mass spectrometry;

PMF: Peptide mass fingerprinting;

PSMD: 26S proteasome non-ATPase regulatory subunit;

SDS-PAGE: Sodium dodecyl sulphate polyacrylamide gel electrophoresis.

Introduction

As an intestinal parasite in chickens, Eimeria tenella invades and rapidly multiplies within host cells to cause severe damage to intestinal mucosa, triggering disease manifestations that are collectively known as avian coccidiosis [7]. Multiple pathogenic Eimeria species, including E. tenella, E. necatrix and E. maxima, are prevalent worldwide [2], to the extent that co-infections with multiple Eimeria species are actually more common than single-species infection [1, 17]. Thus, developing a vaccine that provides cross-protective immunity against multiple pathogenic Eimeria species is desirable.

Currently, anticoccidial drugs and live attenuated vaccines have proved to be effective in preventing coccidiosis, but concerns about drug resistance and drug residues, as well as the potential reversal of vaccine strains to the wild-type (virulent) forms are already widespread in the field [8, 23]. Alternative prevention and control measures, such as DNA vaccines and recombinant subunit protein vaccines, have become the focus of ongoing coccidiosis prevention research [9, 15, 33].

Immunoproteomics has been successfully utilized to screen antigens at different developmental stages of chicken Eimeria and other parasites of veterinary importance. For example, Udonsom et al. [30] identified specific immunoreactive proteins of Neospora caninum sporozoites recognized by sera from cattle infected with various parasites using two-dimensional gel electrophoresis (2-DE) combined with immunoblotting and LC-MS/MS. A total of 20 specific antigen spots corresponding to 14 different antigen proteins were identified among 70 immunogens. Similarly, Qu et al. [29] used 2-DE coupled with Western blotting to systematically screen E. necatrix sporozoite proteins, identifying 98 distinct protein spots exhibiting cross-reactivity with species-specific hyperimmune serum derived from E. necatrix among a total of 680 protein spots resolved. Liu et al. [21] analyzed 620 soluble proteins from E. acervulina sporozoites and detected 21 conserved antigens that could be simultaneously recognized by hyperimmune sera raised against three Eimeria species. These findings indicate the existence of immunogenic and conserved antigens among different Eimeria species and justify a further refinement of feasible vaccine-targets shared by chicken Eimeria.

The key to developing an effective recombinant vaccine against chicken Eimeria lies not only in the quantity of antigens, but also in the selection of optimal and conserved immunogens that can effectively induce protective immune responses [5, 16]. By our own analyses, it is now evident that nearly two dozen E. tenella sporozoite antigens that are recognized by hyperimmune sera against E. tenella, E. necatrix, and E. maxima can serve as a good starting point for the design of a recombinant vaccine targeting all three common Eimeria species.

Materials and methods

Ethical statement

All animal research protocols for this study were approved by the Animal Ethics Committee of Jiangxi Agricultural University (JXAULL2022-025). All experiments were conducted following explicit guidelines of the Experimental Animal Committee under the Ministry of Agriculture and Rural Affairs in China.

Parasites

San Huang chicks were kept for 14 days in a coccidia-free environment before oral inoculation with 2 × 104 sporulated E. tenella oocysts. From day 6 to day 8 post-infection, fecal samples were collected for harvesting oocysts using a saturated saline flotation method [11]. The oocysts were incubated in 2.5% (w/v) potassium dichromate solution at 29 °C for 96 h before storage at 4 °C. Sporulated oocysts were mechanically disrupted using a tissue homogenizer: an equal volume of glass beads to the oocyst pellet was added, and the mixture was processed at 4 °C with a frequency of 70 Hz (10 s operation followed by 10 s pause, repeated for 35 cycles) until the excystation rate reached ≥ 80%. The resulting sporocysts were purified using 50% Percoll gradient centrifugation and then treated with 10% (v/v) San Huang chicken bile and 0.75% (w/v) trypsin at 41 °C for 1 h to fully release sporozoites. After further centrifugation and filtration through a 1,400-mesh sieve, the sporozoites were stored in liquid nitrogen for subsequent use [3].

Sporozoite proteins

Following the established Liu et al. protocol [21], purified E. tenella sporozoites were suspended in cell lysis buffer containing 8M urea. The suspension was subjected to sonication on ice bath to lyse cells and release soluble proteins. After centrifugation at 15,000× rpm for 10 min at 4 °C, the supernatant containing soluble protein was collected and processed with a 2-D clean-up kit. Protein concentration was quantified with a PlusOneTM 2-D Quant Kit (Cytiva, Marlborough, MA, USA).

Isoelectric focusing (IEF)

IEF was performed as previously described [21, 22]. Briefly, E. tenella sporozoite proteins were rehydrated in IPG buffer, incubated for 1 h at room temperature, and centrifuged. Then, 200 μg of protein was loaded onto 24 cm non-linear pH 3–10 IPG strips (Cytiva). Electrophoresis was performed using a PROTEAN IEF cell (Bio-Rad, Hercules, CA, USA) with four-step voltage: S1 (0–50 V, 12 h), S2 (50–8,000 V, 4 h), S3 (8,000–10,000 V, 4 h), and S4 (10,000 V, 4 h). The strips were immediately used for 2-DE.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‑PAGE)

Before SDS-PAGE, each gel strip was incubated for 15 min in equilibration buffer I and II respectively, following the previously described method [21]. Subsequently, SDS-PAGE was performed using 12.5% polyacrylamide gels on the Ettan DALTtwelve system (Cytiva). For Western blotting and silver staining, two separate gels were run simultaneously. The electrophoresis procedure was as follows: apply 3 W/gel was for 45 min first, then increase to 15 W/gel until the tracking dye reached the bottom of the gels. The temperature during electrophoresis was maintained at 16 °C. Gel staining was carried out following the method of Zhu et al. [38], and the gels were imaged using the ArtixScan 1010 Plus (Microtek International, Inc., Hsinchu, Taiwan).

Digital imaging analysis

The 2-DE gels were analyzed using ImageMasterTM 2D Platinum Software (Version 5.0, Cytiva) for spot detection, quantification, as well as comparative and statistical analyses [21].

Immune sera

Using a previously described method [36], 14-day-old San Huang chicks were randomly divided into four groups (n = 25), namely, experimental groups infected with E. tenella, E. necatrix, E. maxima and a negative control group without infection. Each group was kept separately in chicken coops to prevent cross-contamination. For the three experimental groups, each bird was infected with 5 × 104 sporulated oocysts of the corresponding Eimeria species at two weeks of age, and infections were repeated with 5 × 103 sporulated oocysts every three days (on days 3, 6, 9, and 12) to generate hyperimmune sera, while birds in the negative control group received sham inoculation (with distilled water) only. Five weeks after the last inoculation, blood samples were obtained via cardiac puncture, and serum antibody titers were determined by ELISA. Samples with titers greater than 1:1,280 were pooled, aliquoted and stored at −20 °C until use for immunoassays, including Western blot.

Western blotting

In order to obtain uniform protein blotting, each 2-DE gel was cut into four equal parts for transfer. Resulting proteins were transferred to PVDF membranes (Cytiva) following Qu et al. [29], membranes were blocked with 5% skim milk in PBST (PBS, pH 7.4, 0.05% Tween 20) for 2 h at room temperature. Three sera, diluted 1:100 in PBST, were incubated with the membranes at room temperature for 2 h, using serum as a negative control. After washing the membranes three times with PBST, membranes were incubated with goat anti-chicken IgG-HRP (1:2,000; Proteintech Group, Inc., Rosemont, IL, USA) for 2 h at 37 °C. Washed again with PBST for 1 h, membranes were developed with a chemiluminescence kit. Imaging and analysis were done on a ChemiDocTM XRS + with Image LabTM software (Bio-Rad) [32].

MS analysis and database searches

Protein spots were subjected to MS analysis at the experimental center of Nanjing Medical University, using a MALDI-TOF/TOF instrument (Bruker Daltonics, Bremen, Germany). The resulting mass fingerprinting (PMF) data were acquired and analyzed using the Mascot search engine (https://www.matrixscience.com). The parameters for protein retrieval were as follows: 100 ppm mass accuracy, one missed trypsin cleavage site allowed, fixed modifications of carbamidomethyl (C), variable modifications of oxidation (M), 100 parts per million mass accuracy, and MS/MS fragment tolerance set to 0.4 Da. A positive protein search result was determined by a Mascot score of more than 71 (p < 0.05), amino acid sequence coverage greater than 15%, and at least 4 matching peptides [31]. Prediction of non-classical secretion pathways, signal peptides, and transmembrane structures for the positively identified proteins detected by MALDI-TOF/TOF relied on three online servers, i.e., SecretomeP-2.0a Server (https://services.healthtech.dtu.dk/services/SecretomeP-2.0/), SignalP-5.0 Server (https://services.healthtech.dtu.dk/services/SignalP-5.0/), and TMHMM Server v. 2.0 (https://services.healthtech.dtu.dk/services/TMHMM-2.0/) were used.

Results

Proteins revealed by two-dimensional gel electrophoresis (2-DE)

In silver-stained 2-DE gels, 650 protein spots were detected in the total E. tenella sporozoite protein extract, with molecular weights primarily ranging from 10 kDa to 170 kDa (Fig. 1).

thumbnail Figure 1

Eimeria tenella sporozoites proteins revealed by silver staining of two-dimensional polyacrylamide gel. Approximately 650 spots were detected, primarily between 10–170 kDa and pI 3–10.

Detection of cross-reactive immunogens by Western blot

In Western blot assays using hyperimmune sera against three different Eimeria species, 151 proteins were recognized by anti-E. tenella sera, 84 by anti-E. necatrix sera, and 103 by anti-E. maxima sera. Among these readily identified immunogens, 18 E. tenella sporozoite proteins were reactive with all three hyperimmune sera (Figs. 2–4).

thumbnail Figure 2

Western blot using Eimeria tenella sporozoite proteins and hyperimmune serum raised against E. tenella. In all, 151 spots were recognized by anti-E. tenella serum, including 18 that were reactive to immune sera against two other Eimeria species (Figs. 3–4).

thumbnail Figure 3

Western blot using Eimeria tenella sporozoite proteins and hyperimmune serum raised against E. maxima. A total of 103 spots were recognized by anti-E. maxima serum, including 18 that were reactive to immune sera against two other Eimeria species (Figs. 2 and 4).

thumbnail Figure 4

Western blot using Eimeria tenella sporozoite proteins and hyperimmune serum raised against E. necatrix. A total of 84 spots were recognized by anti-E. necatrix serum, including 18 that were reactive to immune sera against two other Eimeria species (Figs. 2–3).

Identities of cross-reactive immunogens

By MALDI-TOF/MS analysis, the 18 conserved immunogens in E. tenella sporozoites could be classified into five, non-overlapping categories based on enzymatic activities and motor functions (Table 1). In sequence alignment and structural analysis, these E. tenella sporozoite proteins showed a broad range of homology with two other related species: from 71.1% to 98.8% with E. necatrix and from 37.9% to 87.5% with E. maxima (Table 2). Based on BLAST searches, four proteins showed over 80% amino acid sequence identity across all three Eimeria species of interest. In terms of functional pathways, using the SecretomeP – 2.0a server, SignalP – 5.0 server, and TMHMM server v2.0 to analyze the non-classical secretory property (SecP), signal peptide (SP), and transmembrane domain (TM), respectively, seven were found to have high SecP scores (>0.6), while four others had transmembrane domains. These proteins almost universally lack signal peptides and are predicted to participate in intercellular signaling (as receptors or signaling molecules) (Table 1).

Table 1

Immunoproteomic and mass-spectrometric identification of Eimeria tenella sporozoite proteins recognized by hyperimmune sera raised against three Eimeria species of interest.

Table 2

Protein amino acid sequence homology among three common Eimeria species of interest.

Discussion

Our study extends previous work by focusing on three main Eimeria pathogens [21]. Using immunoproteomics techniques and mass spectrometry, our research uncovered 18 evolutionarily conserved immunogens in E. tenella sporozoites that are cross-reactive with hyperimmune sera raised against E. tenella and two other related species. These proteins were assigned to diverse and complex functions, including protein translation and transport, motility, and enzymatic activity. Of note, the amino acid sequences of four immunodominant immunogens (protein spots #03, #09, #12, and #14) share over 80% similarity among three Eimeria species. Among these, protein #03 is predicted to have both a signal peptide and transmembrane region, suggesting extracellular secretion or into specific organelles via the classical secretion pathway and possible participation in intercellular communication and related functions. In contrast, the remaining 17 proteins all lack a signal peptide, and the SecP scores of 7 proteins exceed 0.6, possibly localized in specific intracellular compartments or metabolic processes [26]. Presence of transmembrane domains in three other immunogens (proteins #01, #04, and #12) may potentially facilitate ready recognition by the immune system and offer targets for direct interactions with immune effector molecules [14].

In terms of optimal candidates for a multi-species Eimeria vaccine, sequence homology is of top priority, while SecP scores (e.g., > 0.6) or transmembrane presence for surface accessibility and proven immunogenicity are also critical. For example, one translation initiation factor (protein #17) has been shown to induce tumor necrosis factor-alpha (TNF-α) production and inhibit the growth of Toxoplasma gondii [10]. One Zinc finger protein (protein #18) is known to regulate E. tenella gene expression, leading to reduced cecal lesions and oocyst output in infected chickens [37]. Direct targeting of these essential proteins, as well as several transmembrane candidates cited above, is worth further downstream evaluation.

Conserved immunogens with enzymatic activities include an ATP-binding cassette (ABC) protein (protein #01), an adenylate kinase (protein #09), a purine nucleoside phosphorylase (protein #11), and a vacuolar ATPase synthase subunit d (protein #14). The ABC transporter subfamily B member 2 (ABCB2) is involved in the transport of various substrates [4, 19]. Vacuolar ATPase is a highly conserved proton pump that maintains cellular energy balance and regulates organelle pH [25], while purine nucleoside phosphorylase is essential in purine metabolism [27]. The importance of these conserved immunogens is expected to go beyond vaccination, as they could also serve as therapeutic targets.

Among the motility-related proteins, a kinesin motor domain-containing protein (protein #05) has also been shown to have immune protective effects by stimulating the proliferation of peripheral mononuclear cells and the production of IgG2 antibodies [12]. The ATP-dependent metalloprotease FtsH (protein #08) degrades misfolded/excess proteins, which has been shown to be crucial for the infectivity and in vitro growth of Borrelia burgdorferi, the causative agent of Lyme disease [6]. Studies have also shown that the 26S proteasome non-ATPase regulatory subunit (PSMD, protein #16) may enhance immune cell functions [20]. Endonuclease/exonuclease/phosphatase domain-containing proteins (protein #04), as key immune regulators, participate in immune cell DNA repair, immune response modulation, and immune cell apoptosis [24, 34]. However, other proteins in this functional group have limited information to infer their potential as suitable vaccine candidates.

Overall, our study is consistent with earlier observations that conserved Eimeria immunogens not only exhibit multiple functions for various pathways, but also demonstrate potent antigenicity in immunoassays [13, 28]. For proteins involved in multiple essential functions, immune targeting should be suitable for different developmental stages [18, 35]. To this end, our findings here build on previous studies on Eimeria and related immunogens by identifying highly cross-reactive proteins in sporozoites, differing from earlier work that dealt with immunogens like cross-reactive microneme proteins from E. acervulina, E. maxima, and E. mitis sporozoites [35], while also extending Liu et al.’s analysis of conserved antigens from E. acervulina, E. tenella, and E. necatrix sporozoites [22]. The proteins with predicted enzymatic functions or involved in translation offer broad, intracellular targets for immune and other interventions, as seen with multi-epitope DNA vaccines [9], which should supplement existing strategies that focus on antibody protection induced by recombinant antigens [33] or live attenuated vaccines [23].

Of note, the hyperimmune sera used in this study may overestimate immunogenicity, and follow-up studies are needed to validate these using immune sera from naturally infected birds. Likewise, while bioinformatics predictions provide a basis for screening protein functions, they are unable to replace functional assays (e.g., gene knockdowns) as the gold standard for direct verification.

Conclusions

Immunoproteomics is an effective way of identifying conserved immunogens with broad, cross-reactivity across three Eimeria species of veterinary importance. Our findings can serve as a solid foundation for the design and clinical evaluation of novel, next-generation vaccines for combating mixed infections, especially if additional immunoassays (e.g., ELISA and immunization trials) are used to validate the immunogenicity of individual immunogens or their subunits. Further verification using protective immune sera from naturally infected hosts against recombinant immunogens should also benefit such follow-up efforts.

#

Weiyi Chen and Tao Han contributed equally to this paper and should be considered as co-first authors.

Acknowledgments

We thank the members of the Experimental Center of Nanjing Medical University for providing mass spectrometry equipment and their assistance with various experiments; we are also grateful Dr. Charles Li for in-depth discussions about our research project and the presentation of this manuscript.

Funding

This study was supported by funds from (i) the Key Research and Development Project of Jiangxi Province (Grant No. 20203BBF63019), (ii) the Foundation of Key Laboratory of Livestock Infectious Diseases in Northeast China (Shenyang Agricultural University), the Ministry of Education (Grant No. FKLID-2021-01), and (iii) the National Natural Science Foundation of China (Grant No. 31560691).

Conflicts of interest

All authors declare that they have no conflict of interest.

Author contributions statement

LL: experiments, data analysis, and draft manuscript preparation. WC, TH, FS, HZ, XW, and HG: experiments. WC, LL, and TH: data analysis and manuscript preparation. All authors read and approved the final manuscript for publication.

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Cite this article as: Chen W, Han T, Song F, Zhang H, Wang X, Gong H & Liu L. 2025. Identification of highly cross-reactive immunogens in Eimeria tenella sporozoites. Parasite 32, 76. https://doi.org/10.1051/parasite/2025071.

All Tables

Table 1

Immunoproteomic and mass-spectrometric identification of Eimeria tenella sporozoite proteins recognized by hyperimmune sera raised against three Eimeria species of interest.

In the text
Table 2

Protein amino acid sequence homology among three common Eimeria species of interest.

In the text

All Figures

thumbnail Figure 1

Eimeria tenella sporozoites proteins revealed by silver staining of two-dimensional polyacrylamide gel. Approximately 650 spots were detected, primarily between 10–170 kDa and pI 3–10.
In the text
thumbnail Figure 2

Western blot using Eimeria tenella sporozoite proteins and hyperimmune serum raised against E. tenella. In all, 151 spots were recognized by anti-E. tenella serum, including 18 that were reactive to immune sera against two other Eimeria species (Figs. 3–4).
In the text
thumbnail Figure 3

Western blot using Eimeria tenella sporozoite proteins and hyperimmune serum raised against E. maxima. A total of 103 spots were recognized by anti-E. maxima serum, including 18 that were reactive to immune sera against two other Eimeria species (Figs. 2 and 4).
In the text
thumbnail Figure 4

Western blot using Eimeria tenella sporozoite proteins and hyperimmune serum raised against E. necatrix. A total of 84 spots were recognized by anti-E. necatrix serum, including 18 that were reactive to immune sera against two other Eimeria species (Figs. 2–3).
In the text

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