From the Macro to the Micro: Gel Mapping to Differentiate between Sporozoites of Two Immunologically Distinct Strains of Eimeria maxima (Strains M6 and Guelph)

Two immunologically distinct strains of E. maxima were examined in this study: the M6 strain and the Guelph strain. The differential expression between the sporozoites of the two strains of E. maxima was determined by image analysis of 100 μg of protein from each strain separated by standard one- and conventional two-dimensional polyacrylamide gel electrophoresis. In addition to differences in both molecular weight and the electrophoretic mobility, differences in the intensity of polypeptide bands for example, GS 136.4 and M6 169 were explored. Pooled gels were prepared from each strain. A representative 2D-PAGE gel spanning a non-linear pH range of 3–10 of E. maxima strain M6 consisted of approximately 694 polypeptide spots with about 67 (9.6%) of the polypeptide spots being unique relative to the other strain. E. maxima strain GS had about 696 discernable polypeptide spots with 69 spots (9.9%) that differed from those of the M6 strain. In-depth characterization of the variable polypeptide spots; unique polypeptide spots (absence or presence) and shared polypeptide spots with modifications may lead to novel vaccine target in the form of multi-component, multi-stage, multi-immunovariant strains, multi-species subunit vaccine, and diagnostic probe for E. maxima.


Introduction
Eimeria species are obligate intracellular apicomplexan protistan parasites. They are the major cause of chicken coccidiosis, a disease that leads to economic losses in livestock industries, particularly poultry due to intensive rearing conditions. Diarrhea, weight loss, haemorrhage, general weakness, and death are some of the severe clinical signs of infection. The life cycle of chickens were randomly allocated into two groups (i.e. M6-and GS-infected groups) each consisting of 10 birds in a separate room to avoid cross-contamination. The chickens were housed in isolators with external dimensions (2200 Ã 850 Ã 1800mm) for the entire experiment, with food and tap water provided ad libitum. Chickens were exposed to a 12 h/12 h light/darkness regimen at 25°c. The chickens were not abstained from food and water before CO 2 euthanasia. All efforts were made to minimize animal suffering.

Parasite strains
Two strains of E. maxima were compared in this study.
E. maxima Guelph strain (GS). E. maxima GS is a single oocyst-derived strain of E. maxima that was originally isolated from litter samples obtained from a commercial broiler house in Ontario in 1973. Since the initial isolation and subsequent single oocyst cloning, the strain has been maintained at the Ontario Veterinary College, Guelph, Canada by passage through SPF chickens as required. The strain has been stored for longer periods as cryopreserved sporocysts held in liquid nitrogen.
E. maxima M6. This single sporocyst-derived (i.e. genetically clonal) strain of E. maxima was generated from E. maxima; Florida strain (FL) [12]. E. maxima FL was isolated from litter samples during the mid-1990's from a commercial broiler house in Florida, USA.

Parasite propagation and sporozoite isolation
Oocyst propagation. Parasites were propagated in specific pathogen free (SPF) Barred Rock Chickens (Shaver Strain) according to experimental requirements. Sporulated oocysts were inoculated into SPF chickens by oral gavage, and feces were collected at 7-9 days postinoculation from which oocysts were isolated by fecal flotation [13]. Briefly, feces containing unsporulated oocysts were mixed thoroughly with saturated sodium chloride (aqueous) in a blender. The mixture was poured through a 1.5 mm mesh size screen to remove large pieces of debris and the filtrate was centrifuged at 1500×g for 15 min. The pellet was decanted, the top layer containing oocysts was diluted at least 10-fold in distilled water, centrifuged as before and the resulting pellet was resuspended in 2.5% potassium dichromate (w/v, aqueous). The partially purified oocysts were sporulated by agitation in Erlenmeyer flasks covered with a perforated lid on a rotary shaker at 26°C for approximately 5 days. Sporulated oocysts required for sporozoite isolation were stored at 4°C for no more than 4 weeks or used within 6 months as inoculum for further vivo propagation in SPF chickens.
Sporozoite isolation. Sporozoites destined for protein analyses were purified from surface sterilized, sporulated oocysts as follows. Partially purified sporulated oocysts (see above) were pelleted by centrifugation and resuspended in 4 to 5 volumes of ice-cold household bleach (sodium hypochlorite,~5% w/v aqueous) and placed on ice for 10 minutes with occasional shaking. A small amount of distilled water (1-2 ml) was carefully layered onto the top of the oocysts suspended in bleach and the tube was loaded into a swinging bucket centrifuge without agitation and centrifuged at 1500×g for 15 min without rotor braking. The upper ¾ of the contents of the centrifuge tube was collected and diluted at least 10-fold in phosphate-buffered saline (PBS; pH 7.2). The oocysts were washed by repeated centrifugation and resuspension in PBS until traces of bleach odor were removed. The oocyst walls of these highly purified oocysts were broken using a Mickle disintegrator (Brinkman, Westbury, New York), and the sporocysts were isolated by filtration through Nitex™ screen-printing cloth with a 15μm pore size. Excystation of the purified sporocysts found in the filtrate was accomplished by incubation in PBS containing 5% (v/v) chicken bile and 0.25% (w/v) porcine trypsin (Sigma-Aldrich). After excystation, freed viable sporozoites were passed through a 6μm Nitex™ screen-printing cloth [14]. To remove the excystation solution, the sporozoites from the same batch of the samples were washed repeatedly in PBS (pH 7.2) or Tris buffer (pH 9.6) as required. The sporozoites were pelleted by centrifugation, and excess buffer was removed by a micropipette. The sporozoite pellets were kept aliquots at -80°C for experimental use.
Sporozoite preparation for one-and two-dimensional gel electrophoresis The protein preparation was performed according to [10]. Protein was prepared by rapid freezing and thawing 3 times using liquid nitrogen to disrupt sporozoites in lysis buffer containing 7 M urea, 2 M Thiourea, 4%(w/v) CHAPS, 1%(w/v) DTT, 1 mM PMSF and 0.5%(v/v) IPG buffer pH (3-10) dissolved in 40 mM Tris-base pH 9.6. The concentration of protein was determined using a 2-D Quant Kit (Amersham, Uppsala, Sweden) and used at a concentration of 100μg for the one-and two-dimensional gel electrophoresis.
One-dimensional polyacrylamide gel electrophoresis. The protein in lysis buffer was mixed with loading buffer (0.5 M Tris-HCl, pH 6.8, 20% (V/V) Glycerol, 10% SDS, 10% β-mercaptoethanol, 80μl% (V/V) Pyronin (2mg/ml) at a ratio 1:1 and then boiled for 5 minutes before loading. The protein was separated using gradient gels 5% to 20% according to the manufacturer's instructions (BioRad, CA, USA) at a constant current of 30mA for 30 min followed by 50mA for the remainder of the electrophoresis. The running gels were stained using 0.1% Coomassie brilliant blue R-250 in 50% methanol and 10% glacial acetic acid for 30-60 min, and then destained in 10% and 7% glacial acetic acid.
Gels were fixed in 10% (v/v) methanol and 7% (v/v) glacial acetic acid for 1 hr prior to staining with Coomassie brilliant blue R-250 [15]; Silver stain as described by [16] or SYBRO Ruby according to the manufacturer's instructions (Bio-Rad). High-resolution digital images of Coomassie Blue or Silver-stained gels were obtained using a scanner (Agfa, USA). Fluorescent images were obtained using a DyNA Light UV transilluminator (Labnet International Inc. Woodbridge, NJ, USA). Two-dimensional SDS-PAGE gel analyses were accomplished with the aid of PDQuest TM 2-D Analysis Software (BIO-RAD).
As far as maximal reproducibility of 2D-PAGE protein profiles is concerned, the same stock and working solutions, protocols for sample preparation, and downstream procedures for the entire study were employed. Furthermore, simultaneous electrophoresis of batches of gels was performed exploiting multiple, vertical 2D-PAGE systems. All experiments were performed in triplicate. Prior to carrying out comparisons between different gels, polypeptide spots were normalized using the previous software. The density of polypeptide spots was expressed as the mean of three independent gels ± standard deviation (SD).
"In-gel" tryptic digestion and peptide extraction. Tryptic in-gel digestion was carried out as described earlier [17,18] with some modifications: selected polypeptide band from 3 replicate gels was excised from stained 1-D gels. Briefly, Coomassie-stained polypeptide band was homogenized, destained, and washed with 100 mM ammonium bicarbonate, 100 mM ammonium bicarbonate/50% acetonitrile, and 100% acetonitrile in a consecutive manner. The isolated polypeptides were then dried and trypsinized in 50 μL of a 12 ng/μl sequence-grade modified porcine trypsin (Promega, Madison, USA) in 50 mM ammonium bicarbonate, pH 8.6 at 37°C overnight. The extracted tryptic peptide mixtures were concentrated and purified using a Speedvac and C18 Zip Tips according to manufacturer's instructions (Millipore, Billerica, Massachusetts, USA) in corresponding order, and analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (PerSeptive Biosystems, Framingham, Massachusetts, USA).

Calculation of Genetic Distance based on Parasite Strain Proteomes
The genetic distance between the two strains was determined according to [2,19].
Formula used: where D M6, GS = the genetic distance between E. maxima strain GS and M6; N M6, GS = the number of polypeptide spots shared by strain GS and M6 of E. maxima; N M6 = the number of polypeptide spots in strain M6 of E. maxima; N GS = the number of polypeptide spots in strain GS of E. maxima.

One-dimensional polyacrylamide gel electrophoresis
The polypeptide band comparison between the sporozoites of E. maxima strains: GS and M6 exploiting PBS-lysis buffer (pH 7.2). Coomassie blue-stained polypeptide bands of E. maxima GS and E.maxima M6 sporozoites separated by 1D-SDS-PAGE are set out in Fig 1. The Coomassie blue stained gel of E. maxima M6 sporozoite showed remarkably similar collection of polypeptides over the broad range of molecular weights with the following exceptions: polypeptide bands M6 40.7 and M6 67.7 kDa were absent or barely detectable in E. maxima strain GS (Fig 1). Furthermore, E. maxima GS bands; GS 117.5 is absent in E. maxima strain M6. However, GS 55.7 and GS 44.8 kDa were highly detectable in E. maxima; GS strain compared with those of E. maxima strain M6.
The polypeptide band comparison between E. maxima strains: GS and M6 sporozoites employing Tris-lysis buffer (pH 9.6). The polypeptide bands of E. maxima strains: GS and M6 sporozoites were separated on the basis of molecular weights using 5-20% gradient SDS-PAGE. The polypeptide bands consistently showed some differences in the electrophoretic mobility after Coomassie blue staining. The first dimensional-SDS-PAGE staining pattern disclosed that the GS 274.6 and GS 107 polypeptide bands had a faster electrophoretic mobility than M6 282.7 and M6 110.1 respectively (Fig 2). It was also shown that the polypeptide bands; GS 136.4 and M6 169 were intensely stained utilizing Coomassie blue stain.
Mass spectrometric identification of proteins. The separation of complex protein mixtures by one-dimensional polyacrylamide gel electrophoresis (1-D PAGE) in combination with MALDI-TOF analysis followed by peptide mass database searches was used for protein identification. The in-gel digestion and MADI-TOF analysis for the identification of M6 272.5 band is outlined in (Fig 3). The results, as shown in Fig 3, involve the list of masses obtained from M6 272.5 band digest served as the focal point for identifying and characterizing primary structural features in the M6 272.5 band. Experimentally determined masses were used with MASCOT peptide mass fingerprinting (PMF) database searching to match theoretical peptide masses and to predict M6 272.5 band identities (Fig 3). 46 peptides were used for a database search, which identified the M6 272.5 band as TFP250 with 88 score.
The nominal mass (Mr) and the calculated pI were 262860 and 4.19 respectively. The polypeptide spot comparison between E. maxima strains: GS and M6 sporozoites. The large-scale of the E. maxima strains: M6 and GS proteome was characterized by twodimensional polyacrylamide gel electrophoresis (2D-PAGE). The sporozoite fractionation of E. maxima strains: M6 and GS proteins was done using the immobilized pH gradient (IPG) 3-10 non-linear gel strip according to the isoelectric point(1D), and then the sporozoite polypeptide spots were resolved according to their molecular weights (2D). The relative sensitivity of 2D-PAGE was determined after staining of the polypeptide spots resolved on the gels by different staining solutions. A small number of polypeptide spots in the case of Coomassie brilliant blue R-250 and Silver compared to SYPRO Ruby stain (Fig 4A-4C). The number of separated polypeptide spots of E. maxima strains sporozoite proteins were detected by SYBRO Ruby over the pH range of 3-10 nonlinear is shown in Table 1.

Genetic divergence between the two strains GS and M6 of E. maxima
Based on the polypeptide spots identified in paired 2-D gels stained with SYPRO Ruby, the genetic distance was calculated as follows: Thus, D M6, GS is a measure of the genetic distance between the two strains with numbers approaching zero indicating decreasing distance between organisms. Therefore, the genetic similarity is: 1-D M6, GS = 1-0.1782438 = 0.8217562.
The genetic distance is nearer to zero indicating that the close resemblance of the two strains of the same species. The variable polypeptide spots could be divided according to the following flow chart as shown in Fig 5 and the illustrated Figs 6 and 7A-7D. The results of over-expression of the same polypeptide spots are shown in Fig 7E. By comparing the two strains GS and M6 of E. maxima, the unique polypeptide spots (present in one strain and not in the other strain) can be identified based upon the following criteria: isoelectric point that covers the wide range of pH 3-10, molecular weight and the relative intensities (Tables 2 and 3).

Discussion
This study pertains to analysis of the protein profile of two immunologically distinct strains of E. maxima by 2D gel electrophoresis. Although not observed in previous 1-D SDS-PAGE analyses [12], this study was able to distinguish between the sporozoite proteins of two strains of E. maxima GS and M6 utilizing 1-D SDS-PAGE. In particular, polypeptide bands; GS 117.5 was not apparent in the protein profile of E. maxima M6 lysed using a pH 7.2 lysis buffer. Polypeptide bands; M6 40.7 and M6 67.7 kDa were absent or barely detectable in E. maxima GS using the same lysis buffer. However, GS 55.7 and GS 44.8 kDa were highly detectable in E. maxima; GS strain compared with that of E. maxima M6 using macro-scale SDS-PAGE technique and pH 7.2 as a lysis buffer. By using the more alkaline lysis buffer (Tris pH 9.6); this study was able to detect some more subtle differences in the electrophoretic mobility between the strains such as bands GS 274.6 and GS 107 demonstrating a faster electrical mobility than M6 282.7 and M6 110.1 bands, respectively. The electrophoretic mobility between the two aforementioned strains was observed: GS 274.6 polypeptide band has a faster electrical mobility than M6 282.7. The excised band (i.e. M6 272.5) were digested by trypsin and then the tryptic digest was analysed with Matrix-assisted laser desorption ionization time of flight (MALDI-TOF) analysis of the M6 272.5 band tentatively identified this polypeptide band as a thrombospondin-related adhesive protein (TRAP)-like molecule that had been characterized previously from different strain of Eimeria maxima (EmTFP250; Eimeria maxima TRAP Family Protein 250 kDa). The findings of the current study are consistent with those of [20,21] who detected the same molecule; EmTFP250 of the asexual stage antigen of Eimeria maxima. The difference in the molecular weight between the two bands (i.e. GS 267.1 and M6 272.5) that was detected at 1-D SDS-PAGE level may be due to differences in the primary sequence of the molecules in the N-terminal regions of the ectodomain of the molecule and/or post-translational modifications to one or either of these molecules (unpublished data). Therefore, we are forced to elaborate the findings of this study into vaccine which comprises TRAP polypeptides, CD 154 polypeptide, and Salmonella entritidis. We accredit the reader to our published patent for detailed information [22].
In addition to differences in both molecular weight and the electrophoretic mobility, this study explored differences in the intensity of polypeptide bands as a measure of differences in the expressed proteins of these two strains of E. maxima. For example, polypeptide bands; GS 136.4 and M6 169 were intensely stained with Coomassie blue stain. This results contrast with that of [12] who mentioned that no differences were observed by 1-D SDS-PAGE. The close proximity of molecular weight bands that bear the same charge and the possibility for the presence of more than one different polypeptide in the bands in the electrophoretic profile of the two strains resolved by 1-D SDS-PAGE technique led to the use of 2D SDS-PAGE with its intrinsically higher resolving power. This ability to separate proteins on the basis of two different parameters (i.e. charge and molecular weight) was exploited first by [1]. Sensitive visualization of polypeptide spots over a wide linear range is a crucial step for quantification of gene expression at the protein level. Comparison of the stained gels by different stains showed that the SYPRO Ruby fluorescent stain was the more sensitive stain in detection of polypeptide spots than Coomassie blue and Silver stain as had been observed previously. SYPRO Ruby is more convenient, reproducible and an endpoint procedure. This result is in agreement with Annotated polypeptide spot nos. using arrows and M r of the two-dimensional reference maps of E. maxima M6 and Guelph strains using Tris-lysis buffer (pH 9.6). The total sporozoite proteins were resolved by isoelectric focusing (IEF) and separated across the pH range 3-10, 12.5% acrylamide gel. The polypeptide spots were visualized with SYPRO Ruby stain.
doi:10.1371/journal.pone.0143232.g006 [17] who reported that Silver stain was not an end point. By combining a large format 2-D electropherogram together with a more sensitive stain, this study was able to finally demonstrate phenotypic differences between these two immunovariant strains that can potentially explain the lack of cross-reactivity between these parasites in immunocompetent chickens. By combining high resolution 2-D SDS-PAGE with sophisticated image analysis; differences between the strains both in the presence and absence of spots (67unique spots in M6 and an additional 69 unique spots in GS), as well as in the relative expression of various polypeptide spots shared by both strains were detected. In addition to the differences in the level of some polypeptide spots expression (i.e. high expression level versus low level of expression) which is probably due to codon bias; the presence of several copies of the gene that is responsible for the gene product or the presence of mixture of different polypeptides; the sporozoite 2D electropherogram of the E. maxima strains obtained from a consensus polypeptide array of around 10 gels displayed a large number of the variable polypeptide spots. This result is consistent with [2] who was able to produce fingerprint maps to differentiate between the 7 species of Eimeria. The variable polypeptide spots could be divided into unique (presence/absence) and shared polypeptide spots. The latter could be categorized into shared with and without post-translational modifications. The post-translational modifications could be further sorted into move up or move down, such as GS 29.3 and M6 29 or shift to left or to the right, such as GS 20 and M6 19.8.
Of 696 E. maxima M6 and 694 E. maxima GS polypeptide spots resolved by two dimensional polyacrylamide gel electrophoresis and analyzed by PDQuest TM 2-D Analysis Software; only a few differences 69 and 67 unique polypeptide spots were detected for E. maxima M6 and 694 E. maxima GS respectively. Relatedly, a high-resolution 2-DE gel separation over pH ranges 4-7 and 3-10 was also exploited to resolve 460 and 600 polypeptides spots from Eimeria Exploring the Differences between Two Eimeria maxima Strains Using 2D tenella sporozoite using 4-7 and 3-10 pH strips respectively [3], and in a separate study, a total of 845 polypeptides spots in Eimeria tenella sporozoite proteome was identified [23].
A reasonable approach to tackle the lackability of cross-species and even cross-strain protection could be to hypothesize the following hypotheses to unravel this limitation: 1. Shared modified-polypeptide spots in the pair of E. maxima strain The selection pressure elicited by the host on the parasite leads to the emergence of shared modified polypeptide spots, for example GS P20 and M6 P19.8 appear to be post-translation modification and are probably not related to genomic changes (i.e. at the level of mRNA). According to this hypothesis, these shared modified-polypeptide spots GS P20 and P19.8, could represent the business end of an invasive stage or hidden antigens that are used to evade Exploring the Differences between Two Eimeria maxima Strains Using 2D the host immune system prior to establishment of the parasite or to evade the immune system after establishment, respectively. Mass spectrometry and N-terminal amino acid sequence should be done to determine the sequence of these interesting molecules so that their likely identity can be determined.
2. In relation to the spots that are present in one line and not in the other, the protective capability of the unique spots (i.e. presence/absence) in the immunovariant lines can be tested by the following approach: 2.1. Generation of attenuated lines (generally an abbreviated life cycle that results from deletion of one or more asexual endogenous cycles) from the two parent lines.