Introduction

Parasites of the genus Eimeria are the most ubiquitous and economically important parasite of chickens. Every chicken in a production system is considered to be infected with one or more Eimeria species and economic losses are estimated to be over 1 billion dollars annually (Allen and Fetterer 2002). Control of avian coccidiosis is currently accomplished by either medication of feed with anti-coccidial drugs or administration of live vaccines composed of low doses of Eimeria oocysts. The increasing incidence of drug-resistance and cost of live vaccines is prompting alternative control strategies, such as immunization of chickens with recombinant Eimeria proteins.

There have been several attempts to identify and characterize transcripts in developmental stages of Eimeria maxima, Eimeria tenella, and Eimeria acervulina (Wan et al. 1999; Ng et al. 2002; Li et al. 2003; Miska et al. 2004, 2008: Schwarz et al. 2010). In addition, a number of proteins primarily from developmental stages of E. tenella have been identified and characterized (Lal et al. 2009) but there remain a large number of genes or proteins that have been deposited in GenBank whose function is unknown. The genes or proteins that lack an apparent function and appear to be unique to Eimeria may be the most relevant ones to investigate. Unique genes and their products could prove useful as highly specific targets for recombinant vaccine development.

Recently, a transcriptome study of E. maxima, an important pathogenic species, identified a gene with homology to genes in E. tenella and E. acervulina (Schwarz et al. 2010). This gene denoted as Eimeria-specific protein (ESP) codes for a secreted protein with predicted molecular weight of 19 kDa. This gene lacks homology to known gene sequences from other Apicomplexa. Characterization of ESP is important because of its conservation in the three species and its potential as a target for a novel control of Eimeria parasites. In the current study, ESP from E. maxima was further characterized and found to be most highly expressed during the invasive stages of development and associated with the parasite’s rhoptries.

Material and methods

Parasites and hosts

One-day-old Sexsal (Moyers Hatchery, Inc. USA) or broiler (Heritage strain; Longenecker’s Hatchery, Inc. USA) chickens were housed in isolation and given food and water ad libitum. E. maxima (Tyson’s strain) oocysts were maintained by passage through naïve Sexsals 3–4 weeks of age as previously described (Fetterer and Barfield 2003). All animal experiments were approved by the Beltsville Agricultural Research Center Animal Care and Use committee.

For studies of sporulation time-course, unsporulated oocysts collected from the feces of infected birds were incubated with sodium hypochlorite (6 %) on ice for 10 min in twice the volume of the pellet with agitation every 2–3 min. Following bleach treatment, samples were diluted with water and the bleach removed by repeated centrifugation. Oocysts were suspended in PBS containing an antibiotic/antimycotic mixture (GIBCO, USA) and incubated under aeration at 41 °C. At the desired time interval (ranging from 0 to 72 h), an aliquot containing about 1 × 108 oocysts was removed from the incubation flask, centrifuged, and the pellet containing oocysts was resuspended in 1.0 ml of 40 mM Tris and stored at −70 °C.

Sporozoites (SZ) were prepared from sporulated oocysts (less than 30 days post-harvest) as previously described (Fetterer and Barfield 2003) and were purified by filtering through a cellulose filter pad (Fetterer et al. 2004: Fuller and McDougald 1990). Isolated SZ were prepared for microscopy as described below. For merozoites (MZ) collection, 10–20 broiler chicks (2–4 weeks of age) were inoculated with 5 × 105 sporulated oocysts per bird and sacrificed at 96-h post-infection. About 15 cm of the jejunum distal to Meckel’s diverticulum was removed and MZ isolated as previously described (Schwarz et al. 2010). Isolated MZ were pelleted into HBSS and frozen at −70 °C or prepared for microscopy as described below.

Cloning and expression of recombinant E. maxima ESP

RNA extracted from E. maxima sporozoites using TRIzol reagent (Invitrogen, USA) as recommended was treated with two units of DNase then reverse transcribed (Access RT-PCR System; Promega, USA) using the following primers for E. maxima ESP: forward 5′-GTACGGATTTGCTTACAGTCAAG-3′ and reverse 5′-GTCTTGCAGAAGACAGTTGTCTC-3′ under the following conditions: 48 °C for 45 min; 94 °C for 2 min; 40 cycles of 94 °C for 30 s; 53 °C for 60 s; 68 °C for 120 s; and 68 °C for 7 min. The 782-bp ESP amplicon was purified from the agarose gel using a gel extraction kit (Qiagen, USA), cloned into the pGEM-T easy vector system (Promega, USA) and recombinant plasmids were purified with QIAprep Spin Miniprep Kit (Qiagen, USA). Inserts were sequenced using the M13 vector priming sites for confirmation and this sequence for E. maxima ESP has been deposited to GenBank under the accession JN560940.

Another set of primers (ESP_1 forward: 5′-GCAGGATCCATGAAGGGAGCTATCC-3′ and ESP_1 reverse: 5′-GCCGAATTCGTCTATTTACTGCTTGTCCTCAG-3′) were designed and used to amplify a 530-bp region of E. maxima ESP beginning at the predicted methionine start codon through to the predicted stop codon from the plasmid DNA described above under the following thermal cycling conditions: 95 °C 3 min; 30 cycles of 95 °C for 45 s; 60 °C for 30 s, and 72 °C for 60 s; and 72 °C for 7 min. These primers included BamHI and EcoRI restriction sites at the 5′ and 3′ ends, respectively. After gel electrophoresis the E. maxima ESP amplicon was purified (QIAquick Gel Extraction Kit; Qiagen, USA) and ligated into the expression vector system pTrcHis A (Invitrogen) for in frame ligation at the BamHI and EcoRI restriction sites, forming the recombinant E. maxima plasmid rEmESP-pTrcHis. Chemically competent DH5α Escherichia coli were prepared according to standard protocol (Hanahan 1983) and transformed with rEmESP-pTrcHis. Appropriate target and orientation of inserts were confirmed by sequencing using the following flanking vector primers: sense 5′-CTGTACGACGATGACGATAAG-3′; antisense 5′-TCATCCGCCAAAACAGCCAAG-3′. E. coli BL21 (strain optimized for protein expression) (Novagen, USA) was transformed according to manufacturer protocol with the rEmESP-pTrcHis. PCR-confirmed recombinants were cultured at 37 °C in selective media (Luria-Bertani + 100 μg/ml ampicillin) to O.D.600 = 0.6. The expression of rEmESP was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside at 37 °C for 4 h. Cultures were centrifuged at 5,000×g for 10 min at 4 °C, the supernatant decanted and the pellet resuspended at 1:20 of original culture concentration (OC) in native binding buffer (20-mM sodium phosphate; 500-mM sodium chloride; pH 7.8) followed with the addition of phenylmethylsulfonyl fluoride protease inhibitor at 1:10 of the suspension volume and frozen at −70 °C. Three rounds of freeze-thaw using an ethanol dry-ice bath and 37 °C water bath were performed to lyse the cells, which were then treated with DNase and RNase (1:10,000 OC) for 30 min at room temperature and centrifuged for 10 min at 5,000×g. The soluble fraction containing rEmESP was collected and purified using immobilized metal affinity chromatography on a nickel-nitrilotriacetic acid agarose column (Qiagen, USA) according to manufacturer guidelines, visualized using SDS-PAGE and quantified using bicinchoninic acid (BCA) analysis (Thermo Scientific, USA).

Quantitative RT-PCR analysis

Total RNA was isolated from E. maxima as above then treated with 1 U/μg of RQ1 Rnase-free DNase (Promega, USA) prior to first-strand cDNA synthesis using the Advantage® RT-for-PCR Kit with random hexamers for priming (Clontech Laboratories, USA). To compare the relative expression of ESP during E. maxima development, 1 μg of cDNA template was combined with 150 nM forward and reverse primer and 1x EvaGreen Master Mix (Biotium Inc., USA) in 25-μl total volume then real-time PCR amplified on the Mx3000P (Stratagene, USA) using the following cycling conditions: 95 °C for 10 min; 40 cycles of 95 °C for 30 s; 57 °C for 30 s;72 °C for 40 s; and a dissociation curve generated by ramping from 55 to 95 °C.

The transcript levels of two candidate housekeeping genes, 18S rRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were empirically tested for their suitability as normalizing standards in RNA isolated from the developmental stages of E. maxima. Primers designed to amplify the E. maxima GAPDH gene proved to be differentially expressed while primers targeting the small subunit rRNA (18S rRNA) gene (forward 5′-CGGTGAAACTGCGAATGGCTCA-3′ and reverse 5′-GCCTTCCTTAGATGTGGTAGCC-3′) were consistently expressed and thus used as the reference gene to calculate the relative expression level of ESP as: (CTreference − CTtarget). The mean of three technical replicates were calculated and the dataset was transformed to a positive scale in a linear fashion using the following: X = Y + K + 1 where Y is the relative expression level for a sample, K (22.37) is the lowest Y value from the dataset and 1 to plot the lowest value in the dataset at 1 (Schwarz and Evans 2013). A 130-bp region of E. maxima ESP spanning an intron was amplified using forward: 5′-GCTGCTCCTGAGGACATCAG-3′ and reverse: 5′-CCTTCTCCAACTCCTCAGTGCT-3′. These primers were designed to span an intron to avoid amplifying any contaminating gDNA that may have been present following the RNA extraction. Values were expressed as mean + 1 SEM.

Antibody production

Antisera production was performed by a commercial company (Pacific Immunology, USA; Kim et al. 2010). In brief, antisera was generated by a primary immunization with affinity-purified recombinant ESP (rESP) emulsified in Freund’s Complete Adjuvant, followed by two booster immunizations with rESP in Freund’s Incomplete Adjuvant. After the final immunization, sera were collected from rabbits and frozen (−70 °C) in 1-ml aliquots.

Electrophoresis and Western blot analysis

Soluble extracts of E. maxima oocysts were prepared in 40-mM Tris, pH 8.0 containing a cocktail of protease inhibitor DNAse and RNAse as previously described (Fetterer et al. 2004). Samples were incubated for 10 min at 37 °C and then frozen at −70 °C. Concentrations of soluble proteins were measured by BCA assay (Thermo Scientific, USA) with bovine serum albumin as the standard. Samples were analyzed by polyacrylamide gel electrophoresis using 1-mm thick gradient gels (8–9 cm, 4–12 % Bis-Tris, Invitrogen, USA) as described (Fetterer and Barfield 2003). All samples were reduced by addition of 10-mM dithiothreitol. Extracts of sporulated oocysts, SZ and MZ were loaded onto gels at about 10 μg of protein per lane. Western blot analysis was performed using the method previously described (Fetterer and Barfield 2003). Rabbit primary antibody was used in dilutions from 1:500 to 1:1,000. Secondary antibody was goat anti-rabbit conjugated to HRP (20 ng/ml; Thermo Scientific, USA). Chemiluminescence of blots was visualized with a digital camera after exposure to Luminol (SuperSignal West Dura Extend, Thermo Scientific, USA). Western blots were quantified with a gel analysis system (Labworks, UVP, USA).

Antibody localization

For indirect fluorescent antibody staining (IFA) of E. maxima, SZ or MZ were recovered from infected chickens and purified as described above. Parasites were air dried or fixed with methanol or acetone, processed as previously described (Fetterer et al. 2004), and visualized at ×400 magnification with a Zeiss compound microscope. A 1:250 to 1:1,000 dilution of rabbit anti-ESP was used as the primary antibody and 1:100 dilution of FITC-labeled anti-rabbit IgG (Pierce) as a secondary antibody. For antibody localization in infected intestine, tissue was isolated and fixed in 10 % formalin, and 10-μm paraffin sections were prepared (Histoserv Inc., USA). Sections were stained with 1:1000 dilution of rabbit anti-ESP as the primary antibody using Dako (Denmark) Envision with AEC as the substrate chromogen following manufacturer’s recommendation.

For electron microscopy, SZ and MZ isolated as above were fixed for 5 min at room temperature by immersion into 4.0 % paraformaldehyde/0.5 % glutaraldehyde/0.05 M Na-Cacodylate buffer at pH 7.0. This was followed by washing in a 0.1-M Na-Cacodylate buffer rinse, six times over 1 h, dehydrated in an ethanol series and infiltrated with hard-grade acrylic resin (LR White, London Resin Company, UK) and cured at 55 °C for 24 h. The samples were centrifuged at about 1,000×g for 10 min between each solution change. Ninety-nanometer silver–gold sections of the tissue were obtained on a Reichert/AO Ultracut microtome with a Diatome diamond knife and collected onto 200 mesh formvar coated Ni grids. The grids were floated with the tissue section facing down on drops of PBS containing 0.1-M glycine and 1 % BSA for 10 min, washed with PBS, floated on drops of PBS–2 % nonfat dry milk –0.1 % Tween 20 (Tw20), and then floated on drops containing a 1:250 to 1:1,000 dilution of rabbit antiserum in PBS–NFDM–Tw20. Rabbit antiserum to rESP was used in the primary labeling step. The grids were incubated for 2 h at room temperature, washed three times with PBS–NFDM–Tw20, and floated for 1 h at room temperature on drops of a 1:50 dilution of gold particle (10 nm diameter)-labeled goat anti-rabbit IgG (H–L chain specific, Sigma, USA). The grids were washed three times with PBS–Tw20, twice deionized H2O, air dried, stained with 5 % uranyl acetate for 30 min, and examined with a Hitachi H7000 electron microscope.

Results

Quantitative analysis of ESP transcripts in E. maxima

The transcript expression profile of ESP in developmental stages of E. maxima was determined by quantitative RT-PCR. These data were normalized to the level of 18S rRNA from the same template sample and plotted as relative expression (Fig. 1) as described. ESP was transcribed at relatively low levels in oocysts at 0 h, 12 h, and 24-h sporulation with mean relative expression = 1.943 ± 0.489, 2.950 ± 0.085, and 2.003 ± 0.171, respectively. At 48 h, ESP relative expression levels approximately doubled (Fig. 1) and reached cycling threshold ∼8 cycles earlier, increasing mean relative expression levels to 10.107 ± 0.177 in 48-h sporulated oocysts. ESP transcription maintained similar levels through sporulation at 72 h (11.460 ± 0.338) then reached highest levels of relative transcription in SZ (15.430 ± 0.053) and MZ (14.403 ± 0.295).

Fig. 1
figure 1

The relative expression of ESP transcripts from developmental stages of E. maxima. The relative expression of ESP from E. maxima unsporulated oocysts (0 h), sporulating oocysts (12–48 h), sporulated oocysts (72 h), sporozoites (Spz) and merozoites (Mz) were plotted after being normalized to 18SrRNA. Values are means ± SEM; n = 3 technical replicates

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Western blot analysis of ESP in developmental stages

The purified rESP analyzed by gel electrophoresis had a relative molecular weight (Mr) of 24 kDa (Fig. 2a) and stained specifically with the anti-ESP by Western blot (not shown). The theoretical molecular weight (MW) for the entire recombinant predicted translation is 23 kDa while the predicted MW for the native protein is 19 kDa. Western blot analysis of SZ and MZ extracts indicate a single band with Mr of about 23 to 24 kDa recognized by anti-ESP (Fig. 2b)

Fig. 2
figure 2

Gel analysis of recombinant and native ESP. a Gel electrophoresis of recombinant ESP (rESP). About 1 (lane1) or 5 μg (lane 2) was loaded in each lane. Gel was stained with Coomassie blue. Standards (std) have Mr of 21 and 27 kDa. b Western blot analysis of E. maxima MZ lane 1 or SZ lane 2. About 3.0-ug were loaded per lane. Standards (Std) have Mr of 20 and 30 kDa

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Antibody staining

Epifluoresence microscopy of IFA staining of E. maxima SZ indicated that ESP was localized to paired structures in the parasite’s anterior (Fig. 3a). Staining of MZ was also localized to structures in the parasite’s anterior (Fig. 3b). Pre-bleed sera did not react with either SZ or MZ (not shown). Ultrastructural studies demonstrated specific staining of anti-ESP in paired structures in the anterior of the MZ consistent with the morphology of the rhoptries (Fig. 4). In approximate longitudinal sections, the structures appeared elongated (Fig. 4a and c) while in an approximated cross section, the structures are circular (Fig. 4b). In some sections, staining could be observed in membranous structures that were not obviously rhoptries (Fig. 4c). Staining of structures in E. maxima SZ was not observed (not shown).

Fig. 3
figure 3

Immuno-localization by IFA of anti-ESP in E. maxima SZ (a) and MZ (b). The arrows indicate location of anti-ESP stained with fluorescently tagged anti-rabbit IgG

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Fig. 4
figure 4

Ultrastructural localization of anti-ESP in E. maxima MZ. Arrows indicate dark grains that visualize the bound antibody

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Staining of intestine 120 h after infection of birds with E. maxima oocysts demonstrated limited staining of oocysts with staining restricted to outer parasitophorous vacuole (PV) membranes (Fig. 5b). In developing oocysts, weak staining was observed in cytoplasm and PV membranes. Wall-forming bodies were not stained (Fig 5d). General staining of microgametocytes was observed (Fig. 5c) however, staining of macrogametes was not observed (not shown).

Fig. 5
figure 5

Staining of sections of tissue from chicken mid intestine with anti-ESP 120 h after infection with 25,000 oocysts. Positive staining is indicated by dark red color. a Staining with pre-bleed sera. b Oocysts. c Microgametocytes. d Developing oocysts showing wall-forming bodies (blue stain)

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Discussion

Previous studies of the E. maxima transcriptome reported a predicted protein, denominated ESP, which is abundantly expressed in three Eimeria species, is not homologous to other proteins outside the genus Eimeria, and is without known function (Schwarz et al. 2010). The current results extend the knowledge of ESP and indicate it is differentially transcribed throughout development and the protein is associated with both the rhoptries, the PV membranes surrounding the developing oocysts and the microgametes.

Three distinct organelles comprise the apical complex of Apicomplexan parasites: rhoptries, micronemes, and dense granules. All of these organelles are unique to Apicomplexa and play critical roles in mediating host cell invasion (Katz et al. 2006). As a result, proteins produced by these organelles are frequently targeted for vaccine development as a way of minimizing cross-reactivity with host epitopes and for the potential of blocking critical components to host cell invasion. Unlike microneme proteins, which are highly conserved across Apicomplexa (Tomley and Soldati 2001), putative rhoptry proteins are very divergent across taxa (ex. Plasmodium vs. Toxoplasma vs. Eimeria) and may reflect highly specialized coevolution of these parasite proteins with their specific host targets (Boothroyd and Dubremetz 2008). That ESP is unique to Eimeria species is most congruous with our current understanding of rhoptry proteins but not with microneme proteins.

Both light and electron microscopy localized anti-ESP antibody to paired organelles in the anterior of MZ consistent with the structure of the Eimeria rhoptries (Jenkins et al. 1990). The localization of ESP to the rhoptries and potentially other organelles at the apical end of SZ and MZ was the most striking observation and supports a functional role for ESP in mediating host cell interaction. Ultrastructural studies suggest the protein is distributed throughout the rhoptries rather than to specific regions (bulb or neck) as reported for some rhoptry proteins in the related apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii (Rodger et al. 1988; Bradley et al. 2005). In contrast to our results using MZ, no specific staining of SZ by anti-ESP was observed by electron microscopy although light microscopy clearly showed staining in the anterior of the SZ. The reason for this is not apparent. Although it could not be detected by electron microscopy within the SZ and ESP transcripts were found to be highly expressed in SZ by quantitative RT-PCR. Additionally, ESP was detected by Western blot in fully sporulated oocysts, (which contain SZ) as well as isolated SZ.

Although ESP is clearly associated with the rhoptries of SZ and MZ, it was also observed in PV surrounding oocysts and in microgametocytes embedded in host tissue, suggesting important roles beyond initial host cell invasion, possibly maintenance of intracellular stability within the host cell. It is known that some rhoptry proteins from Apicomplexa are secreted during host cell invasion and associate with the PV and PV membrane that forms around the parasite embedded in a host cell (reviewed in Boothroyd and Dubremetz 2008). We have shown that transcription of ESP is active to varying degrees throughout parasite development, congruent with our detection of ESP protein to particular regions of parasites at multiple development stages. Similar to our findings with ESP, previous studies of SZ proteins from Eimeria, such as the microneme protein MIC 2, have increased transcription prior to completion of sporulation and formation of SZ within the oocyst (Sasai et al. 2008; Ryan et al. 2000). A similar pattern of expression would be expected for rhoptry proteins since they are also part of the apical complex. Taken together, these data are consistent with a functional role for ESP as a protein that mediates interaction with the host environment and may also serve as a structural protein of PV membranes and microgametes.

Proteins specific to the rhoptries of the Apicomplexans T. gondii, Neospora caninum, and P. falciparum have been characterized and range in molecular weight from 40–240 kDa (Bradley et al. 2005; Bradley and Sibley 2007; Marugan-Hernandez et al. 2011). Recently, the rhoptry proteome from E. tenella has been characterized (Oakes et al. 2013). A large number of proteins were indentified in the E. tenella rhoptry, many with homology to proteins from T. gondii and N. caninum. Since they did not screen their data against an Eimeria panel of proteins, ESP was not identified because there are no homologues for ESP present in Apicomplexa clades (e.g., Toxoplasma or Neospora) outside of Eimeria as confirmed in Schwarz et al. 2010. Thus, the absence of ESP from these data is misleading. A recently published expressed sequence tag collection produced from E. tenella (Amiruddin et al. 2012) was screened prior to the protein accession of ESP resulting from this study (AEN84770) and therefore did not identify ESP in their data at the time. However, we reexamined their data and indeed found ESP to be present in their “Predicted Secreted Proteins” data file from E. tenella (sequence ID cn1070_Etm102F05) with a BLASTx e-value of 3.00E-63 match to accession AEN84770 (E. maxima ESP). Their analysis of the ESP sequence confirmed the presence of a signal peptide and the lack of transmembrane or GPI anchor regions, consistent with the initial characterization of ESP by Schwarz et al. 2010. In conclusion, this supports that ESP is a secreted protein unique to Eimeria species that localizes to the rhoptry organelles and PV where it most likely mediates host cell interaction.