Vol. 59, No. 3

INFECTION AND IMMUNITY, Mar. 1991, p. 983-989

0019-9567/91/030983-07$02.00/0 Copyright © 1991, American Society for Microbiology

Purification and Characterization of a Protective Antigen from Eimeria tenella YASHWANT D. KARKHANIS,* KARL A. NOLLSTADT, BALBIR S. BHOGAL, OWEN RAVINO, RONALD PELLEGRINO, MARK S. CRANE, P. KEITH MURRAY,t AND MERVYN J. TURNER Department of Biochemical Parasitology, Merck Sharp & Dohme Researcn Laboratories, P.O. Box 2000,

Rahway, New Jersey 07065

Received 23 July 1990/Accepted 20 December 1990

Chickens were partially protected against coccidiosis induced by Eimeria tenella by using extracts prepared from sonicated sporulated oocysts or from sonicated sporozoites. Following gel filtration of either extract, most of the protective antigens were confined to a single pool of proteins in the molecular mass range between 20 and 30 kDa. Further purification of proteins from the protective pool of sporulated oocyst extract yielded a protective polypeptide with a molecular mass of 26 kDa. An antiserum raised to this pool identified a polypeptide with a molecular mass of 22 kDa in the protective pool from a similarly prepared extract of sporozoites of E. tenella. In subsequent studies, this antiserum was used as an aid in cloning protective polypeptides. rpm for 5 min at 4°C in a motorized tissue homogenizer with a loose-fitting pestle. Following centrifugation (600 x g, 10 min, 4°C), the supernatant, a milky, lipid-rich material designated postgrind supernatant, was reserved and the pellet was further processed. Excystation of sporozoites from the released sporocysts in the pellet and purification of sporozoites by passage over DE-52 cellulose, were performed as described previously (13). Preparation of sporulated oocyst antigen (SOA). Sporulated oocysts (50 ml; 2 x 107 ml-1) in PBS containing 1 mM phenylmethylsulfonyl fluoride (Calbiochem-Behring, La Jolla, Calif.) were sonicated with a Branson 340 Sonifier at a power setting of 3 x and a 30% duty cycle. Sonication was performed on ice, and after 5 min the sample was inspected by light microscopy for disruption. Usually, three cycles, each 5 min long, were necessary for complete breakage of the oocysts. The sonicated extract was supplemented with 0.1% (wt/vol) Zwittergent 3-10 (Calbiochem-Behring) and extracted for 18 h at 4°C. Following centrifugation at 27,000 x g for 30 min at 4°C, the supernatant was subjected to gel filtration. Extracts of two different batches of antigen, batches 7 and 11-12, were used separately for gel filtration. Preparation of sporozoite antigens. Sporozoites (109) were washed three times in PBS and sonicated in PBS containing phenylmethylsulfonyl fluoride as described above. The sonicated material was extracted as described above and centrifuged at 100,000 x g for 1 h at 4°C, and the supernatant was used for further testing. Gel filtration. Chromatography of the SOA extract was performed at 4°C on a column (8 by 40 cm) of Sephadex S-200 (Pharmacia Fine Chemicals, Piscataway, N.J.) equilibrated with 50 mM NaH2PO4-Na2HPO4 (pH 7.2)-0.1% Zwittergent 3-10, and the A230 was monitored. Fractions (13 to 14 ml) were collected and pooled according to their sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) patterns. Pooled fractions were dialyzed against 10 liters of 10 mM NH4HCO3 for 96 h with three changes and lyophilized. Lyophilized fractions were stored at -20°C and dissolved in a minimal volume of glass-distilled water prior to in vivo testing. In later experiments, S-200 chromatography was performed in 50 mM NH4HCO3-0.05% Zwittergent

Coccidiosis is a disease of major economic importance to the poultry industry. Control of the six major species of Eimeria is presently accomplished by prophylactic chemotherapy, but the rapid emergence of drug-resistant parasites, coupled with the difficulty and expense of developing new drugs, has led to a search for new approaches to coccidiosis control, such as development of effective vaccines (12). Exposure of chickens to low-grade infections leads to development of species-specific immunity, and several approaches to a safe live vaccine are being pursued. Problems of controlling pathogenicity, coupled with the limited shelf life of such live vaccines, have prompted work toward a subunit vaccine. Most efforts have centered on the use of monoclonal antibodies to identify surface antigens of the sporozoite, since the sporozoite is believed to be the target for protective immunity (4, 5). We report here the results of a more empirical approach, in which the ability of parasite extracts to confer protection was measured and purification of protective antigens was attempted. This approach makes no assumptions about the localization of the protective

antigens. MATERIALS AND METHODS Parasite preparation. Parasites of the Merck LS18 strain of Eimeria tenella were used throughout. Three-week-old chickens (Peterson x Arbor Acre; Avian Services, Frenchtown, N.J.) were infected with 5 x 104 sporulated oocysts. Seven days later, birds were sacrificed, cecal contents were collected, and oocysts, purified by sucrose flotation, were sporulated and sterilized by Clorox treatment as described previously (13). Sporulated oocysts were stored in phosphate-buffered saline (PBS; pH 7.1) at 4°C until further use. A yield of 1 x 107 to 2 x 107 oocysts was obtained from each bird. For preparation of sporozoites, a 2-ml suspension of sporulated oocysts (5 x 107 ml-1) was first ground at 500 * Corresponding author. t Present address: Commonwealth Scientific and Industrial Research Organization Australian Animal Health Laboratory, Geelong, Victoria 3220, Australia.

983

984

KARKHANIS ET AL.

3-10, which affected neither the elution profile nor the protective efficacy but did reduce dialysis time. Additional filtration on a column (1 by 48 cm) of Sephadex G-75 (Pharmacia) was performed in 50 mM ammonium bicarbonate (pH 7.7) without Zwittergent. Fractions were pooled, concentrated by lyophilization, and redissolved in water prior to in vivo testing. Gel filtration columns were calibrated with standard proteins. SDS-PAGE was performed by using a 10% running gel and a 3% spacer gel (8). Gels were stained with either 0.25% Coomassie blue R-250 dissolved in methanol-acetic acid-water (45:10:45, vol/vol) or silver nitrate (10). Protein determination was done as described by Lowry with bovine serum albumin as the standard (9). Carbohydrate was detected by using the phenolsulfuric acid technique (6). Total lipid content was determined by phosphate analysis (2). To obtain the lipid profile, lyophilized preparations (200 pLg of protein) were extracted with chloroform-methanol (2:1) for 1 h on ice with frequent vortex mixing. After extraction, the mixture was filtered through a Bio-Rad Pre-disc (0.45-pLm pore size) and dried under nitrogen. The dried samples were dissolved in chloroform-methanol (2:1; 100 [LI) and analyzed by thin-layer chromatography on silica (Analtech, Inc., Newark, Del.) in methanol. Lipids were detected with iodine vapor (3) and sulfuric acid (3), carbohydrates were detected by anisaldehyde (14), and peptides were detected with ninhydrin in acetone (1). Mobilities were compared with those of phospholipid standards (PL Biochemicals, Inc., Milwaukee,

Wis.). Preparation of antisera. Female New Zealand White rabbits were multiply immunized with fraction V protein. The primary immunization was given in Freund complete adjuvant, with boosts at monthly intervals in Freund incomplete adjuvant. The immunogen was prepared by emulsifying protein (50 p.g) in 0.5 ml of PBS with 0.5 ml of adjuvant, and the entire dose was administered subcutaneously in multiple sites on a shaven patch of the back. Animals were bled for serum after 6 weeks following the primary immunization and then at approximately monthly intervals. Testing of antigens in vivo. Testing of antigens was performed as described previously. Briefly, 2-day-old broiler chicks (Petersen x Arbor Acre) in groups of eight were each immunized with protein (10 p.g) on alum administered intramuscularly in the thigh. Birds received boosts with protein (10 ,ug) on alum on days 9 and 16 and were challenged with oocysts administered by gavage on day 23. The challenge dose was predetermined by oocyst titration to yield a mean cecal lesion score of about 3.5 in nonvaccinated controls. On day 30, birds were sacrificed and lesion scores were assessed by the method of Johnson and Reid (7). The cage model used has been in use for several years. It has been shown that on the basis of treatment group size and variability within the model, a difference of .0.6 in the mean lesion score between treatment groups is statistically significant (11). Western blots (immunoblots) of polypeptide. After SDSPAGE, the slab gel was equilibrated for 30 min in 500 ml of transfer buffer made of 25 mM Tris, 192 mM glycine, 20% (vol/vol) methanol, and 0.1% SDS (pH 8.3). Polypeptides in the gel were transferred electrophoretically to nitrocellulose paper (BA85; 0.4 F.M; Schleicher & Schull, Inc.) in a Transblot transfer cell (Bio-Rad Laboratories). Electrophoresis was done with transfer buffer at 4°C for 16 to 22 h at a constant voltage of 25 V. After transfer, the nitrocellulose paper containing the polypeptides was washed three times, for 20 min each time, with 100 ml of PBS. Excess binding sites on the nitrocellulose paper were blocked by washing

INFECT. IMMUN.

the paper with 100 ml of PBS containing 0.5% (wt/vol) gelatin for 1 h. The nitrocellulose paper was further washed for 30 min in TEN buffer consisting of 50 mM Tris, 0.15 M NaCl, 5 mM EDTA, 0.25% gelatin, 0.5% Triton X-100, and

0.002% sodium azide. The blot was then reacted with 100 ml of TEN buffer containing 0.5 ml of rabbit anti-fraction V serum (dilution, 1:200) for 2 h at room temperature. The blot was washed four times, for 10 min each time, with 80 ml of TEN buffer. The sheet was then exposed to 30 ml of TEN buffer containing [1251]protein A (8,000 to 12,000 cpmI100 p.l; Pharmacia). The blot was washed four times with TEN buffer as described above. After drying, the blot was exposed to Kodak X-Omat AR film and a DuPont Cronex Lightning-Plus intensifying screen (DuPont) for development for 24 h. RESULTS S-200 chromatography. Figure 1 shows the results of S-200 chromatography of the Zwittergent extract of SOA. Several peaks were obtained as revealed by A230. These peaks were divided into several fractions represented by the bars in Fig. 1. However, when these fractions were monitored for protein content, the elution profile was slightly different, which suggests the presence of components other than protein in the extract. SDS-PAGE. Figure 2 shows the SDS-PAGE pattern of the fractions collected from S-200 column chromatography. The molecular masses of the polypeptides ranged from 200 kDa in fraction I to 12 kDa in fraction VI. Since fractions VII and VIII contained low-molecular-weight components, they were not analyzed on SDS-PAGE. Some early-eluting fractions also showed the presence of low-molecular-weight polypeptides. This is perhaps due to their linkage to a polypeptide complex which dissociated under denaturing conditions. In addition to different staining characteristics of polypeptides due to primary structure, the lipid and carbohydrate contents of each fraction affect the staining intensities of the various bands (see Fig. 7). Protective activity against coccidiosis. When these fractions were tested for the ability to protect against coccidiosis induced by E. tenella, fraction V from the various antigen batches tested was consistently active (Fig. 3). Although other fractions of antigen batch 7 showed some in vivo activity, the protection was not as consistent as that shown by fraction V. In some instances, the activities of these other fractions decreased during storage. At other times, their activities could have been due to contamination with fraction V polypeptides. Fractions VII and VIII of SOA extract from batch 7 and fractions VI, VII, and VIII of batch 11-12 were not tested in this group of fractions because they contained low-molecular-weight components and were processed differently. Fraction II of batch 11-12 was lost during processing. However, this fraction obtained from several other batches was consistently inactive. Figure 4 shows a titration of the in vivo protective activities of sporozoite extract, SOA, and fraction V. Both sporozoite extract and fraction V induced partial protection at a dose of 1 p.g per chick, while the SOA extract was protective at doses of .10 p.g per chick. G-75 chromatography. Fraction V was further chromatographed on a G-75 column which yielded three peaks which were divided into four fractions (Fig. 5). Only fraction VB protected chickens against severe coccidiosis (Fig. 6). On SDS-PAGE, this fraction showed a single predominant polypeptide with a molecular mass of 26 kDa with minor bands

VOL. 59, 1991

PROTECTIVE ANTIGEN AND IMMUNITY TO E. TENELLA

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Fraction Number FIG. 1. S-200 chromatography of SOA extract at 4°C (43 mg). The column (8 by 40 cm) was equilibrated with 50 mM phosphate buffer (pH 7.2) containing 0.1% Zwittergent. The flow rate was 75 ml/h. Each fraction was 13 to 14 ml in volume. The bars show different fractions. Fractions I to VI were dialyzed against 10 mM ammonium bicarbonate for 96 h with three changes. They were lyophilized and tested for in vivo efficacy against coccidiosis induced by E. tenella. BSA, Bovine serum albumin.

with lower molecular masses. (Fig. 7). Fraction VA contained several polypeptides with high molecular masses; fraction Vc consisted of low-molecular-mass material, while fraction VD contained salt, since no peptides were seen by

SDS-PAGE. Fraction VB was rechromatographed on the same column several times to obtain the 26-kDa polypeptide (Fig. 8), which was confirmed as a protective antigen in chickens (data not shown).

Lipid analysis. Fraction V from S-200 chromatography of SOA extract contained four different lipids; one migrated slowly and three migrated very fast near the solvent front (Fig. 9), and all stained positively for carbohydrate (Fig. 9, lane D) with anisaldehyde. A similar pattern was observed in a different solvent (chloroform-methanol-acetic acid-water,

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FIG. 2. SDS-PAGE of SOA extract (50 ±g) and fractions I to VI (25 ,ug) of S-200 chromatography (Fig. 1). The gel was stained with Coomassie blue. Lanes: 1, molecular weight standard mixture; 2, SOA extract; 3, fraction 1; 4, fraction II; 5, fraction III; 6, fraction IV; 7, fraction V; 8, fraction VI. The numbers on the left are Mrs

(103).

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FIG. 3. In vivo activities of different fractions: _, S-200 fractions of SOA extract (batch 7); X, S-200 fractions of SOA extract (batch 11-12); , S-200 fractions of sporozoite extract. Fraction II of batch 11-12 was lost during processing. However, this fraction obtained from other batches was consistently inactive. PC is a sporozoite extract and was always used as a positive control.

KARKHANIS ET AL.

986

INFECT. IMMUN.

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FIG. 6. In vivo activities of G-75 chromatography fractions of fraction V (FR. V.) (Fig. 5). SPZ., Sporozoite extract.

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FIG. 4. Titration of in vivo activities of sporozoite extract ( 1), SOA extract ( I ), and fraction V ( M ).

A B

25:4:1, vol/vol). The observation that all fractions (I to VI) of S-200 showed identical lipid profiles, indicated that the protection observed with fraction V was not due to the detectable lipid content. Ninhydrin-positive material was not present in any of the S-200 fractions following extraction for lipid analysis, except for the starting SOA Zwittergent extract (lane C). In summary, overall chemical analysis of fraction V showed that it consisted of 87 to 90% protein, 7 to 10% carbohydrate, and 2 to 3% lipid.

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Fraction Number FIG. 5. G-75 chromatography of fraction V (from S-200 chromatography [Fig. 1]). A 1-mg sample of fraction V dissolved in 50 mM ammonium bicarbonate was applied to the column (1 by 48 cm). The flow rate was 20 ml/h. The bars show different fractions. The column was calibrated with human transferrin (Mr, 80,000), ovalbumin (Mr, 45,000), chymotrypsinogen (Mr, 25,500), and RNase (Mr, 13,700).

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VOL. 59, 1991

987

PROTECTIVE ANTIGEN AND IMMUNITY TO E. TENELLA -

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Sporozoite extract. Figure 10 shows the S-200 elution pattern of the sporozoite Zwittergent extract (100,000 x g supernatant). This pattern was different from that obtained from SOA extract (Fig. 1), presumably because of different starting materials of both of the extracts, as shown by SDS-PAGE patterns (Fig. 2 and 11). Fractions were selected arbitrarily and could not be compared with SOA fractions in terms of Kay and Ve/Vo; only fractions V of both of the extracts were comparable in terms of these two parameters. K0y and VeIVt of fraction V of the sporozoite extract were 0.32 and 0.55, respectively, while those of fraction V of the SOA extract were 0.39 and 0.59, respectively. Fraction V of the sporozoite extract was protective (Fig. 3) and contained few polypeptides. Fractions IV, VI, and VII (data for this fraction not shown in Fig. 3) were also protective and contained a common 22-kDa polypeptide which was not present in nonprotective fractions I to III (Fig. 11). Western blot. Protective fractions from SOA (fraction V) and sporozoite extract (fractions IV and V) were transferred to nitrocellulose paper and immunoblotted with rabbit antifraction V (SOA) serum (Fig. 12). Although several polypeptides were present in fractions from both extracts, the predominant antigenic polypeptide in SOA was a 26-kDa species while that in the sporozoite extract was 22 kDa (Fig. 12, arrowheads). Although there were other peptides in the sporozoite extract which reacted with rabbit antifraction V serum, these polypeptides were also present in nonprotective S-200 fractions I to III.

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A B C D E I FIG. 9. A typical lipid profile of SOA extract and S-200 chromatography fraction. For details, see Materials and Methods. GL1, GL2, GL3, and GL4, glycolipids 1 to 4, respectively; P1, P2, and P3 peptides, ninhydrin-positive spots; ZW, Zwittergent; S, sphingomyelin; PC, phosphatidylcholine; PI, phosphatidylinositol; PS, phosphatidylserine; PE, phosphatidylethanolamine; SF, solvent front. Lanes: A, PBS; B, Zwittergent; C, SOA extract; D, fraction V from S-200 chromatography of SOA extract (Fig. 1); E, phospholipid mixture.

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DISCUSSION From the Zwittergent extract of the sporulated oocysts of E. tenella, we obtained a fraction which partially protected chickens against coccidiosis induced by E. tenella. Cecal lesions, evaluated by the method of Johnson and Reid (7), are rated on a scale of 0 (no pathologic changes, normal gross morphology) to 4 (most severe pathologic changes) on the basis of subjective observation. Although this is a linear scale, the degrees of pathologic change represented by the scale are not linear. Lesion scores of less than 2 represent mild coccidiosis which would not have a serious effect on

111

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FIG. 10. S-200 chromatography of sporozoite extract (100,000 x g supernatant, 20 mg) at 40C. The column (2.5 by 87 cm) was equilibrated with 50 mM ammonium bicarbonate (pH 7.7) containing 0.05% Zwittergent. The flow rate was 30 ml/h. Each fraction volume was 2.4 ml. Fractions in tubes 86 to 110 showing no A230 were pooled and designated fraction X.

988

KARKHANIS ET AL.

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broiler performance. In contrast, lesion scores of 2 or greater represent severe disease which would affect broiler performance considerably. Thus, the reduction in lesion score from a mean of greater than 3 to a mean of less than 2 induced by immunization with fraction V represents a substantial degree of protection. During S-200 column chromatography, this fraction eluted near myoglobin (25.5 kDa) and contained several polypeptides. Upon further fractionation on a G-75 column, a protective 26-kDa polypeptide was obtained. When an antiserum against fraction V was used to identify the antigenic polypeptides in SOA extract and in

INFECT. IMMUN.

fraction V by Western blot analysis, this polypeptide showed a strong reaction. This result was achieved with preparations of fraction V obtained from several batches of SOA. This polypeptide can also be obtained by affinity chromatography. When fraction V of SOA was applied to an affinity matrix, Reactigel (Pierce, Rockford, Ill.), to which immunoglobulin G from rabbit anti-fraction V serum was conjugated, only the 26-kDa polypeptide bound to the column and was eluted with 0.2 M glycine hydrochloride (pH 2.3). This polypeptide, after dialysis against 0.1 M phosphate buffer (pH 7.0), reacted strongly with rabbit anti-fraction V serum on a Western blot and protected chickens against E. tenella (data not shown). It was also possible to isolate this polypeptide by extraction of SOA extract or fraction V with n-butanol to eliminate lipids, carbohydrates, and many proteins. The 26-kDa polypeptide partitioned into the aqueous layer and reacted with rabbit anti-fraction V serum on a Western blot (data not shown). However, both of these procedures produced a low yield of the polypeptide and therefore extensive protection studies, like those done with the gel filtered material (Fig. 1, fraction V, or Fig. 7, fraction VB), could not be performed. When a Zwittergent extract from sporozoites of E. tenella was separated by S-200 chromatography, several protective fractions were obtained, including fraction V. However, all of these fractions contained a common 22-kDa polypeptide which reacted strongly with antisera to fraction V of SOA. Because of the limited amount of available material, fraction V obtained from sporozoites could not be processed further to isolate this polypeptide in quantities sufficient for in vivo testing. Chemical analysis of fraction V of SOA showed that it consists of 87 to 90% protein, 7 to 10% carbohydrate, and 2 to 3% lipid. Because of the limited amount of material, we could not establish the role, if any, that the carbohydrate played in the observed protective activity of fraction V. Moreover, it is unlikely that the lipid is involved in such protection, since the lipid profiles of all S-200 fractions were similar on the basis of thin-layer chromatography of chloroform-methanol extracts (Fig. 9). The retention of antigenicity of the 26-kDa polypeptide after n-butanol extraction of fraction V, which

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FIG. 12. SDS-PAGE (A) and Western blot (B) analyses of SOA and sporozoite extracts and protective fractions derived from these extracts by S-200 chromatography. The stain for SDS-PAGE was silver nitrate; the antibody used for the Western blot was rabbit anti-fraction V. The arrowheads show the protective polypeptides in SOA (26 kDa) and in sporozoite extract (22 kDa). Although other antigenic polypeptides were present in S-200 protective fractions of sporozoites (B), they were also present in nonprotective fractions (Fig. 11). Lanes: 1, fraction IV, SOA batch 7; 2, fraction V of SOA batch 11-12 rechromatographed on a G-75 column; 3, SOA batch 28 Zwittergent extract; 4, fraction V from SOA batch 28; 5, subfraction of fraction V from batch 28; 6, Bio-Rad high- and low-molecular-weight standards; 7, sporozoite batch 1 extract; 8, fraction IV, sporozoite batch 1 extract; 9, sporozoite batch 2 extract; 10, fraction IV, sporozoite batch 2 extract; 11, fraction V, sporozoite batch 2; 12, fraction IV, sporozoite batch 3 extract. The numbers to the sides are MrS (103).

PROTECTIVE ANTIGEN AND IMMUNITY TO E. TENELLA

VOL. 59, 1991

removed lipids and carbohydrates, also supports this conclusion. By using two neutralizing antibodies, Brothers et al. (4) have identified a similar 25-kDa polypeptide on the surface of E. tenella sporozoites. However, this antigen consisted of two 17- and 8-kDa polypeptides linked by a disulfide bond; the 26-kDa polypeptide reported by us is a single-chain protein, since its mobility on SDS-PAGE was independent of the presence of 2-mercaptoethanol in the SDS-PAGE solubilization buffer. In addition, Danforth and McAndrew (5) raised hybridoma antibodies against the sporozoite stages of three species of avian coccidia. Five hybridoma antibodies reacted with 22- and 28-kDa antigens or a diffused band in the 7- to 10-kDa range. Two-dimensional gel electrophoresis has indicated that there are several polypeptides in the 26- to 28-kDa range (unpublished data). Thus, identification of particular polypeptides is dependent on sequence data. The difference in the molecular sizes of the protective polypeptides of E. tenella SOA and sporozoites reported here is also noteworthy. The 22-kDa polypeptide in sporozoites is not a processed product of the 26-kDa polypeptide present in SOA, since antibody against recombinant protein blotted predominantly a 26- to 28-kDa polypeptide and a small amount of a 22-kDa polypeptide in sporozoite extract; the 22-kDa polypeptide could be a proteolytic degradation product of the 26-kDa polypeptide. Because of the restricted heterogeneity of fraction V and, therefore, the reactivity of the specific antiserum, we deemed anti-fraction V serum a suitable reagent for screening of a Agtll library of E. tenella with the aim of eventually identifying protective recombinant proteins. The results of this screening exercise will be presented elsewhere. ACKNOWLEDGMENTS

We thank Mark Gnozzio for providing the different batches of sporulated oocysts and sporozoites of E. tenella and Stefan Galuska for preparation of antiserum to fraction V. REFERENCES 1. Bailey, J. L. 1962. Techniques in protein chemistry, Elsevier Publishing Co., New York.

p.

1-36.

989

2. Bartlett, G. R. 1959. Phosphorous assay in column chromatography. J. Biol. Chem. 234:466-468. 3. Bolliger, H. R., M. Brenner, H. Ganshirt, H. K. Mangold, H. Seiler, E. Stahl, and D. Waldi. 1965. In E. Stahl (ed.), Thin layer chromatography, p. 150-151. Academic Press, Inc., New York. 4. Brothers, V. M., I. Kuhn, L. S. Paul, J. D. Gabe, W. H. Andrews, S. R. Sias, M. T. McCamman, E. A. Dragon, and J. G. File. 1988. Characterization of a surface antigen of Eimeria tenella sporozoites and synthesis from a cloned cDNA in Escherichia coli. Mol. Biochem. Parasitol. 28:235-248. 5. Danforth, H. D., and S. J. McAndrew. 1987. Hybridoma antibody characterization of stage-specific and stage-cross-reactive antigens of Eimeria tenella. J. Parasitol. 73:985-992. 6. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350-356. 7. Johnson, J., and W. M. Reid. 1970. Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens. Exp. Parasitol. 28:30-36. 8. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 9. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:969-975. 10. Morrissey, J. H. 1981. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117:307-310. 11. Murray, P. K., B. S. Bhogal, M. S. J. Crane, and T. T. MacDonald. 1986. Eimeria tenella-in vivo immunization studies with sporozoite antigen, p. 564-573. In L. R. McDouglad, L. P. Joyner, and P. L. Long (ed.), Research in avian coccidiosis. University of Georgia, Athens. 12. Rose, M. E. 1982. Host immune responses, p. 329-371. In P. L. Long (ed.), The biology of coccidia. University Park Press, Baltimore, Md. 13. Schmatz, D. M., M. S. J. Crane, and P. K. Murray. 1984. Purification of Eimeria sporozoites by DE-52 anion exchange chromatography. J. Protozool. 31:181-183. 14. Stahl, E., and U. Kaltenbach. 1961. Dunnschichtchromatographie. VI. Mitteilung. Spurenanalyse von Zuckergemischen auf Kiesulgur G-Schichten. J. Chromatogr. 5:351-355.

Purification and characterization of a protective antigen from Eimeria tenella.

Chickens were partially protected against coccidiosis induced by Eimeria tenella by using extracts prepared from sonicated sporulated oocysts or from ...
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