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Eimeria tenella: B-cell epitope mapping following primary and secondary infections
Experimental Parasitology 113 (2006) 235–238 www.elsevier.com/locate/yexpr
Eimeria tenella: B-cell epitope mapping following primary and secondary infections Alireza Talebi a,¤, Grace Mulcahy b a
Poultry Diseases Division, Department of Clinical Sciences, Faculty of Veterinary Medicine, Urmia University, P.O. Box 1177, Urmia, Iran b Department of Veterinary Microbiology and Parasitology, Faculty of Veterinary Medicine, UCD, BelWeld, Dublin 4, Ireland Received 30 June 2005; received in revised form 17 January 2006; accepted 18 January 2006 Available online 28 February 2006
Abstract Immunisation against coccidiosis has become more reliable and eVective with improved administration techniques for new vaccines. On the other hand, an ideal coccidial vaccine should contain both B- and T-cell immunogenic epitopes. Fine speciWcity of B-cell epitopes recognised by antibodies prepared following primary and secondary infections with Eimeria tenella were studied using “PepScan” techniques. Mapping of B-cell epitopes within an antigenic sequence from E. tenella showed that four distinct types of epitopes were recognised by the host immune system during the primary and secondary infections with the parasite. These observations demonstrated that new epitopes are also involved in induction of antibody responses following the secondary infection. © 2006 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Eimeria tenella; Antibody responses; Epitope mapping; PepScan
1. Introduction As the world’s poultry industry continues to expand, so does concern for the control of coccidiosis, where increasing drug-resistance and drug-residue problems threaten to ban chemotherapeutic control of coccidiosis (Dalloul and Lillehoj, 2005). Broad-spectrum immunological prophylaxis is desirable alternative for control of coccidial infections in the poultry industry. Both humoral and cellular responses are involved in resistance to coccidiosis. Occurrence of cross-reactivity among antiEimeria sera (Talebi and Mulcahy, 1995b; Xie et al., 1992) in the absence of resistance against heterologous infections has been reported. Lack of correlation between antibody responses and protection following natural infections (Augustine et al., 1991; Talebi and Mulcahy, 1995a) have been interpreted to mean that cell-mediated immunity plays a major role (Lillehoj et al., 1989; *
Corresponding author. Fax: +98 441 2777099. E-mail addresses: [email protected], [email protected] (A. Talebi), [email protected] (G. Mulcahy). 0014-4894/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2006.01.005
Rose and Wakelin, 1989) in protection. On the other hand, passive protection of chicks by maternally transmitted antibodies (Smith et al., 1994a,b; Wallach et al., 1992) and monoclonal antibodies (Crane et al., 1988; McDougald, 2003; Wallach et al., 1990) have been reported. In vitro studies on the eVectiveness of antibodies in protecting cell cultures from invasion of sporozoites (Larsen et al., 1991; Talebi, 2001) indicate that antibodies could have deleterious eVects on the developmental stages of Eimeria, as reported by Rose (1987). Antibodies might therefore play a role in the prevention of secondary infection with Eimeria species (Parmentier et al., 2001). These observations suggest that quality and amount of key-epitope-speciWc antibodies are more important than the quantity of humoral responses overall. Although the kinetics of antibody responses to coccidial infections have been studied, recognition of key epitopes involved in the development of immunity has not been completely investigated. The aim of this study was to analyse the Wne speciWcity of B-cell epitopes involved in induction of antibody responses following primary and secondary infections with E. tenella.
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2. Materials and methods 2.1. Parasite and inoculation of chickens Groups of one-day-old chicks (Cobb 500) were legbanded, raised in wire cages in isolated coccidia-free conditions, and fed ad libitum on non-medicated diet. At six weeks of age, one group (six chickens) was infected with two doses (at 3 weeks intervals) of 2 £ 104 sporulated oocysts (suspended in 2 ml of distilled water) of E. tenella (Johnson and Long, 1989) via direct injection through the skin into the crop as previously described (Mockett and Rose, 1986). Blood samples were taken from the vena cutanea ulnaris (wing vein) of the chickens on day 0 (before inoculation) and weekly post-inoculation (pi) using 2 ml vacutainer tubes. The sera were collected and stored at ¡20 °C. 2.2. Preparation of oocysts antigens Oocysts of E. tenella were harvested for four days (6–9th day pi), sporulated, puriWed, and stored at 4 °C as described (McDougald, 1978; Oz et al., 1984). Water-soluble antigens were prepared from the puriWed sporulated oocysts (Rose, 1977) and the protein concentration was determined by the bicinchoninic acid technique (Pierce) using a standard curve constructed with bovine serum albumin as internal standard. 2.3. Assessment of antibody levels of the antisera Antibody capture immunoassay (ELISA) was used for determination of antibody levels of the sera. BrieXy, 96-well Nunc microplates were coated with 5 g/ml of oocyst antigen in carbonate/bicarbonate buVer pH 9.6 (100 l/well) and after incubation overnight at room temperature, the coated plates were washed Wve times with PBS containing 0.1% Tween 20 (washing buVer) and dried on tissue paper. Serial threefold dilutions of the sera were made in dilution buVer (1% sodium caseinate, 10% sheep serum, and 0.1% Tween 20 in PBS). Starting at a serum dilution of 1/5, 100 l/well was used. The plates were incubated for 1 h at 37 °C and then washed Wve times to remove unbound antibodies. SpeciWc bounded antibodies were detected by the sequence addition of rabbit anti-chicken horse-radish peroxidase (R HRPO) conjugate (SUN 157, Serotec Ltd., Oxford, England) and the substrate 3,3,5,5’-tetramethylbenzidine (TMB, Sigma Co., England) in PBS (pH 5.0). The substrate reaction was stopped after 10 min by adding 100 l/well of 10% H2SO4. The plates were read in an ELISA microplate reader at 450 nm with 492 nm reference. The results were assessed as OD at serum dilution 1/810. 2.4. B-cell epitope mapping analysis An overlapping hexapeptide synthesised on polypropylene pins, based on an immunogenic sequence of E. tenella
antigen (Talebi and Mulcahy, 1994), was used as a solid phase of the ELISA to screen immunogenic B-cell epitopes by measuring the reactivity of each hexapeptide with chicken antisera obtained after primary and secondary infections. The assays were carried out according to the manufacturer’s (Cambridge Research Biochemicals, Cambridge, UK) instructions. BrieXy, the pins were blocked in 200 l/well pre-coating buVer (10% sheep serum and 2% bovine serum albumin in PBS containing 0.1% Tween 20) for 1 h on a shaking table (100 rpm) at room temperature to block non-speciWc binding sites. After washing Wve times with PBS containing 0.1% Tween 20 (washing buVer), the pins were incubated in 175 l/well antiserum (1/ 2000 dilution) for 1 h, shaking at room temperature, or 24 h at 4 °C. They were washed Wve times with washing buVer to remove non-speciWc bound antibodies, and then incubated in 175 l/well rabbit anti-chicken (IgG) horse-radish peroxidase (SUN 157, Serotec, UK) conjugate at dilution 1/1000 in conjugate buVer (1% sheep serum and 0.1% sodium casein in PBS containing 0.1% Tween 20) for 1 h in the shaking condition at room temperature. After washing Wve times with PBS only, 150 l/well azino-bis-ethylbenzthiazoline sulphonic acid (ABTS) solution [1 ABTS tablet in 20 ml substrate buVer (sodium azide and citric acid in 100 ml of distilled water, pH 4.0)] was added and incubation of the pins continued for 10 to 45 min. The reactivity of each hexapeptide was measured at 415 nm with 492 nm reference absorbance wavelength by reading the last microplate in an ELISA reader (Bio-Rad) and the results were expressed as optical density values for each well corresponding to each hexapeptide of the “PepScan.” 3. Results 3.1. Serum antibody production following primary and secondary infections Antibody responses of infected chickens increased following the primary infection, peaked at 14 days pi, and then declined gradually. The secondary infection boosted the antibody responses of the infected group, while those of control chickens remained unchanged during the experimental period. 3.2. B-cell epitope mapping analysis The speciWc reactivity of antibodies with each hexapeptide is presented in Fig. 1 (day 0 shows non-speciWc reactions of control chickens and before inoculation of infected chickens, and 1 week pi to 5 week pi represent speciWc reaction). Reactions with OD 7 0.7, which were at least three times of OD on day 0, were considered speciWc to the epitope(s) involved. As shown in Fig. 2, only 18 sets of 90 hexapeptides were recognised by the antisera and there were at least four distinct groups of antibodies: (1) antibodies that reacted with AGAGGA epitope were highest during the primary infection and boosted by the secondary
A. Talebi, G. Mulcahy / Experimental Parasitology 113 (2006) 235–238
237
Fig. 2. Epitopes recognised by chicken sera obtained following primary and secondary infections with E. tenella. -䊐-, antibody only in 1 week (GGEQVP), –䉫–, antibody increased following secondary infection (GAGGAG), –䊊–, antibody level following primary infection decreased after secondary infection (AGGAEG), and ––, antibody level boosted following secondary infection (AGAGGA). Fig. 1. Reactivity of sera from infected and control chickens with 90 overlapping hexapeptides, which had been synthesised on polypropylene pins (pin numbers are indicated on the X axis) based on immunogenic sequence of E. tenella antigen. Day 0 (before primary infection), 3 weeks pi (before secondary infection), and 5 weeks pi (end of the experiment). Sera were used in the assay at dilution 1/2000, the dilution being chosen to optimise the signal-to-noise ratio.
infection. (2) Antibodies that reacted with AGGAEG epitope were relatively high during the primary infection but declined during the secondary infection. (3) Antibodies that reacted with GAGGAG epitope did so only slightly during the primary infection but more signiWcantly after the secondary infection. (4) Antibodies that reacted with some epitopes such as GGEQVP only at one occasion (week 4 or 5). 4. Discussion Increasing resistance to a species of Eimeria following repeated exposure to oocysts of homologous species is always presumed to be due to cell-mediated immunity (CMI) and it has been suggested that cytotoxic T-cells (Bhogal et al., 1989), lymphokines (Kogut and Slajchert, 1992), and T-cell dependent antibodies as well as other elements may be involved in protection. The eVects of B-cells
on T-cell immunity (Bhogal et al., 1986, 1987) and eVectiveness of antibodies on sporozoites may suggest that the isotype, availability of the invasion sites and in particular epitope-speciWcity of antibodies are more relevant to protection than the total antibody titres. In the case of speciWcity of antibodies to the key epitopes involved in the invasion process, it has been demonstrated that speciWc antibodies can be detected (Tajima et al., 2003) as well as that epitope-speciWc antibodies can mimic sporozoite antigens and prevent invasion (Bhogal et al., 1988). Synthetic peptide technology provides powerful tools for screening immunogenic epitopes with high-resolution speciWcity and for design of synthetic immunogens containing both beneWcial key B- and T-cells epitopes. For example, Wne speciWcity analysis of antibody responses during this study clearly demonstrated that new epitopes are recognised by the host immune system during repeated infections with E. tenella. 5. Conclusion Recognition of immunogenic epitopes of antigens following repeated exposure to sporulated oocysts of E. tenella diVers in some respect from those epitopes recognised by the host during primary infection. As the degree of
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protection following secondary infection increases, identiWcation of key immunogenic epitopes involved in protective immunity against each species of Eimeria will expedite the development of a vaccine capable of inducing adequate cross-species protection against these parasites. References Augustine, P.C., Danforth, H.D., Barta, J.R., 1991. Development of protective immunity against Eimeria tenella and E. acervulina in white Leghorn chickens inoculated repeatedly with high doses of turkey coccidia. Avian Diseases 35, 535–541. Bhogal, B.S., Jacobson, E.B., Tse, H.Y., Schmatz, D.M., Ravino, O.J., 1989. Parasite exposure elicits a preferential T-cell response involved in protective immunity against Eimeria species in chickens primed by an internal-image anti-idiotope antibody. Infection and Immunity 57, 2804–2810. Bhogal, B.S., Nollstadt, K.H., Karkhanis, Y.D., Schmatz, D.M., Jacobson, E.B., 1988. Anti-idiotopic antibody with potential use as an Eimeria tenella sporozoite surrogate for vaccination of chickens against coccidiosis. Infection and Immunity 56, 1113–1119. Bhogal, B.S., Nollstadt, K.H., Karkhanis, Y.D., Jacobson, E.B., 1987. Induction of antibody and cellular immune responses in chickens against Eimeria tenella by anti-idiotope. In: Weber, W.T., Ewert, D.L.J. (Eds.), Avian immunology, Proceedings of the 2nd International Conference on Avian Immunology (Philadelphia). Progress in Clinical and Biological Research, vol. 238, Alan R. Liss Inc., NY, pp. 307–320. Bhogal, B.S., Tse, H.Y., Jacobson, E.B., Schmatz, D.M., 1986. Chicken T-lymphocyte clones with speciWcity for Eimeria tenella. I. Generation and functional characterization. Journal of Immunology 137, 3318–3325. Crane, M.S.J., Murray, P.K., Gnozzio, M.J., McDonald, T.T., 1988. Passive protection of chickens against Eimeria tenella infection by monoclonal antibody. Infection and Immunity 56, 972–976. Dalloul, R.A., Lillehoj, H.S., 2005. Recent advances in immunomodulation and vaccination strategies against coccidiosis. Avian Diseases 49, 1–8. Johnson, J.K., Long, P.L., 1989. The fecundity and drug sensitivity of precocious lines of avian Eimeria. In: Yvore, P. (Ed.), Coccidia and Intestinal Coccidiomorphs, Proceedings of the Vth International Coccidiosis Conference, Tours (France). INRA Service des Publications, Paris, pp. 671–676. Kogut, M.H., Slajchert, T., 1992. T-lymphocytes confer protection in chickens against Eimeria tenella by production of lymphokines. Immunology and Infectious Diseases 2, 69–79. Larsen, N.C., Rasmussen, K.R., Healey, M.C., 1991. Production and partial characterisation of monoclonal antibodies speciWc for the gamonts of Eimeria tenella. Journal of Parasitology 77, 1012–1015. Lillehoj, H.S., RuV, M.D., Bacon, L.D., Lamont, S.J., JeVers, T.K., 1989. Genetic control of immunity to Eimeria tenella: interaction of MHC genes and non-MHC linked genes inXuences level of disease susceptibility in chickens. Veterinary Immunology and Immunopathology 20, 135–148. McDougald, L.R., 1978. The growth of avian Eimeria in vitro. In: Long, P.L., Boorman, K.N., Freeman, B.M. (Eds.), Avian Coccidiosis, Proceedings of the thirteenth Poultry Science Symposium. British Poultry Science, Ltd, Edinburgh, pp. 185–223.
McDougald, L.R., 2003. Coccidiosis. In: Saif, Y.M. (Editor-in-chief), Diseases of Poultry 11th Edition. Iowa State Press, A Blackwell Publishing Company, Ames, Iowa 50041, pp. 974–991. Mockett, A.P.A., Rose, M.E., 1986. Immune responses to Eimeria: quantiWcation of antibody isotypes to Eimeria tenella in chicken serum and bile by means of the ELISA. Parasite Immunology 8, 481–489. Oz, H.S., Markham, R.J.F., Bemrick, W.J., Stromberg, B.E., 1984. Enzymelinked immunosorbent assay and indirect haemagglutination techniques for measurement of antibody responses of E. tenella in experimentally infected chickens. Journal of Parasitology 70, 859–863. Parmentier, H.K., Abuzeid, S.Y., De Varies- Reilingh, G., Nieuwland, M.G.B., Graat, E.A.M., 2001. Immune responses and resistance to Eimeria acervulina of chickens divergently selected for antibody responses to sheep red blood cells. Poultry Science 80, 894–900. Rose, M.E., 1977. Eimeria tenella: skin hypersensitivity to injected antigen in the fowl. Experimental Parasitology 42, 129–141. Rose, M.E., 1987. Immunity to Eimeria infections. Veterinary Immunology and Immunopathology 17, 333–343. Rose, M.E., Wakelin, D., 1989. Mechanisms of immunity to coccidiosis. In: Yvoure, P. (Ed.), Coccidia and Intestinal Coccidiomorphs, Proceedings of the Vth International Coccidiosis Conference, Tours (France). IRNA Service des Publications, Paris, pp. 527–539. Smith, N.C., Wallach, M., Miller, C.D., Morgenstern, R., Braun, R., Eckert, J., 1994a. Maternal transmission of immunity to Eimeria maxima: enzyme-linked immunosorbent assay analysis of protective antibodies induced by infection. Infection and Immunity 62, 1348–1357. Smith, N.C., Wallach, M., Petracca, M., Braun, R., Eckert, J., 1994b. Maternal transfer of antibodies induced by infection with E. maxima partially protects chickens against challenge with E. tenella. Parasitology 109, 551–557. Tajima, O., Onaga, H., Nakamura, T., 2003. An enzyme-linked immunosorbent assay with the recombinant merozoite protein as antigen for detection of antibodies to Eimeria necatrix. Avian Diseases 47, 309–318. Talebi, A., 2001. Inhibition of Eimeria acervulina sporozoite invasion by rabbit and chicken antisera using ISI assay. Archive Razi Institute (Iran) 52, 49–59. Talebi, A., Mulcahy, G., 1994. High-resolution mapping of B-cell epitopes within an antigenic sequence from Eimeria tenella. Infection and Immunity 62, 4202–4207. Talebi, A., Mulcahy, G., 1995a. Correlation between immune responses and oocyst production of chickens monospeciWcally infected with E. maxima. Avian Pathology 24, 485–495. Talebi, A., Mulcahy, G., 1995b. Cross-reactivity among antisera raised against Wve avian Eimeria species in the natural host and a laboratory animal. Avian Pathology 24, 533–544. Wallach, M., Pillemer, G., Yarus, S., Halabi, A., Pugatsch, T., Mencher, D., 1990. Passive immunisation of chickens against Eimeria maxima infection with a monoclonal antibody developed against a gametocyte antigen. Infection and Immunity 58, 557–562. Wallach, M., Halabi, A., Pillemer, G., Sar-Shalom, O., Mencher, D., Gilad, M., Bendheim, U., Danforth, H.D., Augustine, P.C., 1992. Maternal immunisation with gametocyte antigens as a mean of providing protective immunity against Eimeria maxima in chickens. Infection and Immunity 60, 2036–2039. Xie, M., Gilbert, J.M., McDougald, L.R., 1992. Electrophoretic and immunologic characterisation of proteins of merozoites of Eimeria acervulina, E. maxima, E. necatrix and E. tenella. Journal of Parasitology 78, 82–86.
Eimeria tenella: B-cell epitope mapping following primary and secondary infections Alireza Talebi a,¤, Grace Mulcahy b a
Poultry Diseases Division, Department of Clinical Sciences, Faculty of Veterinary Medicine, Urmia University, P.O. Box 1177, Urmia, Iran b Department of Veterinary Microbiology and Parasitology, Faculty of Veterinary Medicine, UCD, BelWeld, Dublin 4, Ireland Received 30 June 2005; received in revised form 17 January 2006; accepted 18 January 2006 Available online 28 February 2006
Abstract Immunisation against coccidiosis has become more reliable and eVective with improved administration techniques for new vaccines. On the other hand, an ideal coccidial vaccine should contain both B- and T-cell immunogenic epitopes. Fine speciWcity of B-cell epitopes recognised by antibodies prepared following primary and secondary infections with Eimeria tenella were studied using “PepScan” techniques. Mapping of B-cell epitopes within an antigenic sequence from E. tenella showed that four distinct types of epitopes were recognised by the host immune system during the primary and secondary infections with the parasite. These observations demonstrated that new epitopes are also involved in induction of antibody responses following the secondary infection. © 2006 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Eimeria tenella; Antibody responses; Epitope mapping; PepScan
1. Introduction As the world’s poultry industry continues to expand, so does concern for the control of coccidiosis, where increasing drug-resistance and drug-residue problems threaten to ban chemotherapeutic control of coccidiosis (Dalloul and Lillehoj, 2005). Broad-spectrum immunological prophylaxis is desirable alternative for control of coccidial infections in the poultry industry. Both humoral and cellular responses are involved in resistance to coccidiosis. Occurrence of cross-reactivity among antiEimeria sera (Talebi and Mulcahy, 1995b; Xie et al., 1992) in the absence of resistance against heterologous infections has been reported. Lack of correlation between antibody responses and protection following natural infections (Augustine et al., 1991; Talebi and Mulcahy, 1995a) have been interpreted to mean that cell-mediated immunity plays a major role (Lillehoj et al., 1989; *
Corresponding author. Fax: +98 441 2777099. E-mail addresses: [email protected], [email protected] (A. Talebi), [email protected] (G. Mulcahy). 0014-4894/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2006.01.005
Rose and Wakelin, 1989) in protection. On the other hand, passive protection of chicks by maternally transmitted antibodies (Smith et al., 1994a,b; Wallach et al., 1992) and monoclonal antibodies (Crane et al., 1988; McDougald, 2003; Wallach et al., 1990) have been reported. In vitro studies on the eVectiveness of antibodies in protecting cell cultures from invasion of sporozoites (Larsen et al., 1991; Talebi, 2001) indicate that antibodies could have deleterious eVects on the developmental stages of Eimeria, as reported by Rose (1987). Antibodies might therefore play a role in the prevention of secondary infection with Eimeria species (Parmentier et al., 2001). These observations suggest that quality and amount of key-epitope-speciWc antibodies are more important than the quantity of humoral responses overall. Although the kinetics of antibody responses to coccidial infections have been studied, recognition of key epitopes involved in the development of immunity has not been completely investigated. The aim of this study was to analyse the Wne speciWcity of B-cell epitopes involved in induction of antibody responses following primary and secondary infections with E. tenella.
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2. Materials and methods 2.1. Parasite and inoculation of chickens Groups of one-day-old chicks (Cobb 500) were legbanded, raised in wire cages in isolated coccidia-free conditions, and fed ad libitum on non-medicated diet. At six weeks of age, one group (six chickens) was infected with two doses (at 3 weeks intervals) of 2 £ 104 sporulated oocysts (suspended in 2 ml of distilled water) of E. tenella (Johnson and Long, 1989) via direct injection through the skin into the crop as previously described (Mockett and Rose, 1986). Blood samples were taken from the vena cutanea ulnaris (wing vein) of the chickens on day 0 (before inoculation) and weekly post-inoculation (pi) using 2 ml vacutainer tubes. The sera were collected and stored at ¡20 °C. 2.2. Preparation of oocysts antigens Oocysts of E. tenella were harvested for four days (6–9th day pi), sporulated, puriWed, and stored at 4 °C as described (McDougald, 1978; Oz et al., 1984). Water-soluble antigens were prepared from the puriWed sporulated oocysts (Rose, 1977) and the protein concentration was determined by the bicinchoninic acid technique (Pierce) using a standard curve constructed with bovine serum albumin as internal standard. 2.3. Assessment of antibody levels of the antisera Antibody capture immunoassay (ELISA) was used for determination of antibody levels of the sera. BrieXy, 96-well Nunc microplates were coated with 5 g/ml of oocyst antigen in carbonate/bicarbonate buVer pH 9.6 (100 l/well) and after incubation overnight at room temperature, the coated plates were washed Wve times with PBS containing 0.1% Tween 20 (washing buVer) and dried on tissue paper. Serial threefold dilutions of the sera were made in dilution buVer (1% sodium caseinate, 10% sheep serum, and 0.1% Tween 20 in PBS). Starting at a serum dilution of 1/5, 100 l/well was used. The plates were incubated for 1 h at 37 °C and then washed Wve times to remove unbound antibodies. SpeciWc bounded antibodies were detected by the sequence addition of rabbit anti-chicken horse-radish peroxidase (R HRPO) conjugate (SUN 157, Serotec Ltd., Oxford, England) and the substrate 3,3,5,5’-tetramethylbenzidine (TMB, Sigma Co., England) in PBS (pH 5.0). The substrate reaction was stopped after 10 min by adding 100 l/well of 10% H2SO4. The plates were read in an ELISA microplate reader at 450 nm with 492 nm reference. The results were assessed as OD at serum dilution 1/810. 2.4. B-cell epitope mapping analysis An overlapping hexapeptide synthesised on polypropylene pins, based on an immunogenic sequence of E. tenella
antigen (Talebi and Mulcahy, 1994), was used as a solid phase of the ELISA to screen immunogenic B-cell epitopes by measuring the reactivity of each hexapeptide with chicken antisera obtained after primary and secondary infections. The assays were carried out according to the manufacturer’s (Cambridge Research Biochemicals, Cambridge, UK) instructions. BrieXy, the pins were blocked in 200 l/well pre-coating buVer (10% sheep serum and 2% bovine serum albumin in PBS containing 0.1% Tween 20) for 1 h on a shaking table (100 rpm) at room temperature to block non-speciWc binding sites. After washing Wve times with PBS containing 0.1% Tween 20 (washing buVer), the pins were incubated in 175 l/well antiserum (1/ 2000 dilution) for 1 h, shaking at room temperature, or 24 h at 4 °C. They were washed Wve times with washing buVer to remove non-speciWc bound antibodies, and then incubated in 175 l/well rabbit anti-chicken (IgG) horse-radish peroxidase (SUN 157, Serotec, UK) conjugate at dilution 1/1000 in conjugate buVer (1% sheep serum and 0.1% sodium casein in PBS containing 0.1% Tween 20) for 1 h in the shaking condition at room temperature. After washing Wve times with PBS only, 150 l/well azino-bis-ethylbenzthiazoline sulphonic acid (ABTS) solution [1 ABTS tablet in 20 ml substrate buVer (sodium azide and citric acid in 100 ml of distilled water, pH 4.0)] was added and incubation of the pins continued for 10 to 45 min. The reactivity of each hexapeptide was measured at 415 nm with 492 nm reference absorbance wavelength by reading the last microplate in an ELISA reader (Bio-Rad) and the results were expressed as optical density values for each well corresponding to each hexapeptide of the “PepScan.” 3. Results 3.1. Serum antibody production following primary and secondary infections Antibody responses of infected chickens increased following the primary infection, peaked at 14 days pi, and then declined gradually. The secondary infection boosted the antibody responses of the infected group, while those of control chickens remained unchanged during the experimental period. 3.2. B-cell epitope mapping analysis The speciWc reactivity of antibodies with each hexapeptide is presented in Fig. 1 (day 0 shows non-speciWc reactions of control chickens and before inoculation of infected chickens, and 1 week pi to 5 week pi represent speciWc reaction). Reactions with OD 7 0.7, which were at least three times of OD on day 0, were considered speciWc to the epitope(s) involved. As shown in Fig. 2, only 18 sets of 90 hexapeptides were recognised by the antisera and there were at least four distinct groups of antibodies: (1) antibodies that reacted with AGAGGA epitope were highest during the primary infection and boosted by the secondary
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237
Fig. 2. Epitopes recognised by chicken sera obtained following primary and secondary infections with E. tenella. -䊐-, antibody only in 1 week (GGEQVP), –䉫–, antibody increased following secondary infection (GAGGAG), –䊊–, antibody level following primary infection decreased after secondary infection (AGGAEG), and ––, antibody level boosted following secondary infection (AGAGGA). Fig. 1. Reactivity of sera from infected and control chickens with 90 overlapping hexapeptides, which had been synthesised on polypropylene pins (pin numbers are indicated on the X axis) based on immunogenic sequence of E. tenella antigen. Day 0 (before primary infection), 3 weeks pi (before secondary infection), and 5 weeks pi (end of the experiment). Sera were used in the assay at dilution 1/2000, the dilution being chosen to optimise the signal-to-noise ratio.
infection. (2) Antibodies that reacted with AGGAEG epitope were relatively high during the primary infection but declined during the secondary infection. (3) Antibodies that reacted with GAGGAG epitope did so only slightly during the primary infection but more signiWcantly after the secondary infection. (4) Antibodies that reacted with some epitopes such as GGEQVP only at one occasion (week 4 or 5). 4. Discussion Increasing resistance to a species of Eimeria following repeated exposure to oocysts of homologous species is always presumed to be due to cell-mediated immunity (CMI) and it has been suggested that cytotoxic T-cells (Bhogal et al., 1989), lymphokines (Kogut and Slajchert, 1992), and T-cell dependent antibodies as well as other elements may be involved in protection. The eVects of B-cells
on T-cell immunity (Bhogal et al., 1986, 1987) and eVectiveness of antibodies on sporozoites may suggest that the isotype, availability of the invasion sites and in particular epitope-speciWcity of antibodies are more relevant to protection than the total antibody titres. In the case of speciWcity of antibodies to the key epitopes involved in the invasion process, it has been demonstrated that speciWc antibodies can be detected (Tajima et al., 2003) as well as that epitope-speciWc antibodies can mimic sporozoite antigens and prevent invasion (Bhogal et al., 1988). Synthetic peptide technology provides powerful tools for screening immunogenic epitopes with high-resolution speciWcity and for design of synthetic immunogens containing both beneWcial key B- and T-cells epitopes. For example, Wne speciWcity analysis of antibody responses during this study clearly demonstrated that new epitopes are recognised by the host immune system during repeated infections with E. tenella. 5. Conclusion Recognition of immunogenic epitopes of antigens following repeated exposure to sporulated oocysts of E. tenella diVers in some respect from those epitopes recognised by the host during primary infection. As the degree of
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