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Specificity and Crossreactivity of Immune Serum and Hybridoma Antibodies to Various Species of Avian Coccidia
Specificity and Crossreactivity of Immune Serum and Hybridoma Antibodies to Various Species of Avian Coccidia HARRY D. DANFORTH and PATRICIA C. AUGUSTINE US Department of Agriculture, Agricultural Research Service, Animal Parasitology Institute, Beltsville, Maryland 20705 (Received for publication March 4, 1983) ABSTRACT The species-specificity and crossreactivity of serum antibodies (Ab) from birds immunized specifically with six different species of coccidia and of 24 hybridoma antibodies (Ab) developed against four species of chicken and two species of turkey coccidia were determined by use of the indirect immunofluorescent antibody (IFA) test on air-dried sporozoites. With few exceptions, the immune chicken sera were found to crossreact with all species of coccidia tested. Seven of the hybridoma Ab were species-specific, while the other 17 Ab demonstrated varying degrees of crossreactivity. Similar types of IFA patterns were seen with both the species-specific and crossreactive hybridoma Ab. Some of the crossreactive hybridoma Ab produced one type of IFA pattern with the sporozoites against which they were originally raised and different patterns with other species of sporozoites. The development of the hybridoma Ab has made it possible to identify the species of coccidia found in mixed infections and check the purity of laboratory strains. (Key words: avian coccidia, hybridoma antibodies, immune chicken serum, specificity, crossreactivity) 1983 Poultry Science 62:2145-2151 INTRODUCTION T h e specificity of i m m u n i t y t o Eimeria species is well d o c u m e n t e d (see reviews b y Rose, 1 9 7 3 ; 1 9 7 8 ; 1 9 8 2 ) and has been used t o identify species (Rose, 1982) and t o d e t e r m i n e t h e prevalence of various species of avian coccidia in commercial p o u l t r y houses. Strain variation in i m m u n i t y elicited in birds occurs within various coccidial species, and b o t h c o m m o n and specific antigens have been d e m o n s t r a t e d (Rose, 1 9 8 2 ) . These studies primarily used parameters associated with t h e bird, such as weight gain, feed conversions, lesion scores, and o o c y s t s c o u n t s , o r tested for t h e presence of Ab in serum from animals exposed t o a coccidial infection. Danforth ( 1 9 8 2 ) developed t h e t e c h n i q u e s necessary for producing h y b r i d o m a antibodies directed against various species of avian coccidia. T h u s , it is n o w possible t o d e t e r m i n e if individual antibodies are specific only for t h e species of coccidia against which t h e y are p r o d u c e d or if t h e y crossreact w i t h antigenic d e t e r m i n a n t s of o t h e r coccidial species. In this paper w e r e p o r t on t h e p r o d u c t i o n of m o n o c l o n a l antibodies directed against four species of chicken and t w o species of t u r k e y coccidia. We also d e t e r m i n e d and c o m p a r e d t h e species specificity or crossreactivity of i m m u n e
chicken sera and t h e m o n o c l o n a l antibodies o n seven species of chicken and t w o species of t u r k e y coccidia. MATERIALS AND METHODS Immune Chicken Serum (ICS) Production. White Leghorn chickens raised coccidia-free were immunized against t h e following species and strains of coccidia b y giving t h e m six oral inoculations of 2 0 , 0 0 0 o o c y s t s during a 3-week interval: E. acervulina (API No. 12), E. maxima (API No. 4 1 ) , E. mitis (API No. 50), E. brunetti (API N o . 32), E. necatrix (API N o . 4 8 and 52), and E. tenella (API No. 10 and 2 4 ) . T h e birds were bled 7 days after t h e final inoculation, and t h e ICS were recovered and stored in 2-ml aliquots at —80 C until used. Preparation of Sporozoites. Sporozoites of t h e chicken coccidial species E. acervulina (API N o . 12), E. mitis (API N o . 50), E. mivati (API N o . 6), E. maxima (API N o . 4 1 ) , and E. tenella (API N o . 2 4 ) , and t h e t u r k e y species E. adenoeides (API N o . 20) and E. meleagrimitis (API N o . 4) were excysted and cleaned (Doran and Vetterling, 1 9 6 7 , B o n t e m p s and Yvore, 1 9 7 4 ) . These parasites were used t o i m m u n i z e mice for development of h y b r i d o m a cell lines and were also air dried o n i m m u n o f l u o r e s c e n t a n t i b o d y slides.
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Production of Hybridoma Antibodies. The procedures for production of hybridoma antibodies (Ab) against Eimeria species have been previously described (Danforth, 1982). Briefly, Balb/CByJ mice (Jackson Laboratory, Bar Harbor, Maine) were immunized via tail vein injection with sporozoites of the species of chicken and turkey coccidia previously listed. The mice were given three injections of 100,000 viable sporozoites at 2-week intervals and a fourth injection one week later. Spleens were removed from the mice 3 days after the last injection, forced through a 60-mesh screen, mixed at a 10:1 ratio with P3-X63-Ag8 mouse myeloma cells, and fused in the presence of polyethylene glycol (Baker Co., Phillipsburg, NJ) as described by Kennett et al. (1978). The hybridoma cells produced by the fusion procedure were grown in Dulbecco's medium containing hypoxanthine, aminopterin and thymidine in 96-well, flat bottom microtiter plates (Falcon) (Danforth, 1982). Cell culture supernatants from wells with cell growth were screened for Ab production by the indirect immunofluorescent antibody (IFA) test using air-dried sporozoites of the same coccidial species against which the mice were immunized. Fifty out of 600 hybridoma cell lines that produced Ab against various species of sporozoites were then cloned by the limiting dilution technique previously described (Danforth, 1982). Based on the type of immunofluorescent pattern produced on or in air-dried sporozoites, 24 clones (Tables 2 and 3) were chosen to determine species-specificity or crossreactivity with the species and strains of Eimeria previously listed. Staining by Immunofluorescent Antibody. Air-dried sporozoites were first exposed to ICS or cell culture supernatant containing the hybridoma Ab for 30 min at 37 C. The slides were washed for 10 min in phosphate-buffered saline (PBS) at pH 7.3 and then treated for 30 min at 37 C with a pretitrated dilution of fluorescein-conjugated rabbit anti-mouse IgG (Miles Lab, Elkhart, IN). The slides were then washed for 10 min in fresh PBS, mounted with coverslips in the presence of glycerol, and viewed with a Zeiss ultraviolet epifluorescence microscope.
RESULTS
The ICS produced against individual species of avian coccidia were, in most cases, found to
crossreact with the sporozoites of eight chicken and two turkey species of coccidia (Table 1). The exceptions were the E. acervulina (API No. 12) ICS, which did not react with either E. adenoeides or E. meleagrimitis, and the E. maxima (API No. 41) and E. tenella (API No. 24) ICS that did not react with E. mivati sporozoites. Seven of the 24 monoclonal hybridoma antibodies were found to be species-specific (Table 2) and 17 were crossreactive (Table 3). Both the species-specific and the crossreactive antibodies demonstrated four different IFA patterns of tip, surface, refractile body, and internal (Tables 1 and 2; Figs. 1 to 4). In addition, one species-specific antibody directed against E. tenella had an internal anterior V4 IFA pattern, which was not seen with any of the crossreactive monoclonal antibodies (Table 2), and one crossreactive clone produced an IFA pattern that labeled both surface pellicle and internal area of the parasite, which was not observed with any of the species-specific Ab (Table 3). The hybridoma antibodies that labeled the refractile bodies of the sporozoites were usually crossreactive with all species of coccidia tested (Table 3 and Fig. 3). However, one monoclonal Ab directed against E. adenoeides (designated 91-C7) labeled only the refractile body of this species (Table 2). Two other monoclonal Ab, designated 12-27 and 95-G10, that labeled the surface and surface-interior, respectively, also crossreacted with all species of coccidia tested (Table 3). Some of the crossreactive hybridoma Ab demonstrated one pattern with the homologous sporozoites and a still different pattern with sporozoites of other species (Tables 3 and 4). These variations were seen for all IFA patterns except the refractile body, which was area specific, regardless of species. In some cases, there was much variation in IFA patterns. For example, Ab from the 12-27 clone labeled the surface of the homologous E. acervulina clone sporozoites but gave either a tip IFA pattern with E. adenoeides or an internal IFA pattern with E. meleagrimitis (Table 4). Similarly, clone 95—G7, that gave a tip IFA pattern against E. adenoeides, gave a surface-internal pattern with E. mivati and E. maxima sporozoites (Table 4). Other monoclonal Ab that showed either an internal IFA pattern (94-G2) or combination surface-internal pattern (95G10) on the E. adenoeides sporozoites that
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they were directed against, labeled other areas of the heterologous, crossreactive sporozoites (Table 4). Antibody produced against the E. adenoeides turkey species demonstrated greater variation in IFA patterns than Ab produced against any of the chicken species (Table 4). By use of crossreactive and species-specific monoclonal Ab, it is possible to identify and speciate coccidial parasites in mixed infections (Figs. 4-6). A mixed population of coccidia is easily recognized by use of the 12-27 crossreactive Ab (Table 3; Fig. 4), and then speciation of the mixed population, in this example E. tenella and E. acervulina, can be accomplished with the species-specific F8-3B7 and 12-04 hybridoma Ab (Figs. 5 and 6). In this way, it is also possible for laboratory strains maintained at the Poultry Parasitic Diseases Laboratory to be periodically checked for purity.
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This investigation has shown that it is possible to produce both crossreactive and species-specific hybridoma antibodies against various species of avian coccidia. This is similar to hybridoma antibodies directed against other protozoan parasites such as Plasmodium yoelli (Taylor et al, 1981) in which species-specificity and crossreactivity between red blood cell stages was also observed with various hybridoma cell lines. Unlike the sera from birds immunized against a single species of coccidia that crossreacts with other species, some hybridoma Ab are species-specific and can be used to identify coccidia species in mixed infections.
TABLE 2. Species-specific hybridoma
antibodies
Cloned cell line
Speciesspecificity
Immunofluorescent pattern 1
F8-3B7 B6-1H4 12-04 12-23 05-2F11 76-G6 91-C7
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These results also indicate that identification of the species of coccidia present in field litter samples can be quickly made without the use of bird immunization (Levine, 1938, 1942; Moore and Brown, 1951; Long, et al, 1976). Whether strain variations would occur with hybridoma Ab such as was seen with bird immunity to E. acervulina and E. maxima (Rose, 1978; 1982) will require further investigation. However, it has been previously reported that there is no apparent strain specificity seen with speciesspecific hybridoma Ab directed E. tenella sporozoites (Danforth, 1982). Eimeria species elicit specific immunity (Rose, 1973; 1978). Accordingly, there must be some antigen(s) or antigenic determinant(s) specific to single species of coccidia for such immunity to be elicited in birds. Thus, it is to be expected that some of the hybridoma Ab would react with only the species of coccidial sporozoite against which they were raised. Shared antigen or antigenic determinants between coccidial species have also been demonstrated (Rose, 1982). Hence, it was not surprising that Ab directed against crossreactive antigenic determinants were produced in the hybridoma model. It is evident that during the evolutionary development of coccidial parasites, some common antigens were retained or conserved, while others unique to the specific species of coccidia were developed. It is not known why there were different IFA patterns seen on sporozoites exposed to some of the crossreactive hybridoma Ab. The antigenic determinants recognized by the Ab are apparently located in different areas of these sporozoites. Whether this crossreactivity indicates the presence of homologous molecules must await further biochemical analysis. Efforts are now underway to produce hybridoma antibodies directed against the remaining pathogenic species of chicken and turkey coccidia. Preliminary evidence suggests that a diagnostic procedure utilizing an enzyme-linked immunoadsorbent assay (ELISA) could then be used for the diagnosis of species of coccidia in commercial poultry houses.
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2150
DENFORTH AND AUGUSTINE
RESPONSES TO HYBRIDOMA AND COCCIDIAL ANTIGENS
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FIG. 1. Specific tip immunofluorescent antibody (IFA) pattern seen with E. adenoeides sporozoites exposed to a hybridoma antibody (designated 95-G9) that was originally raised against this same species of parasite. X 2000. FIG. 2. Surface IFA pattern seen on E. maxima sporozoites exposed to a hybridoma antibody (designated 12-27), which showed a surface-internal IFA pattern with the E. acervulina sporozoites against which it was originally raised. X 2000. FIG. 3. Refractile body IFA pattern seen with E. adenoeides sporozoites exposed to a hybridoma antibody (designated 91C7) that was originally raised against this species of parasites. Although this particular refractile body hybridoma was species-specific, the other crossreactive refractile body hybridomas demonstrated the same type of IFA pattern. FIG. 4. Surface-internal IFA pattern demonstrated by the 12-27 crossreactive hybridoma when it was exposed to a mixed population of parasites (E. tenella and E. acervulina). X 2000. FIG. 5. General internal IFA pattern seen with an E. tenella species-specific hybridoma antibody (designated F8-3B7) with the same mixed population of sporozoites used in Figure 4. Note that only the E. tenella sporozoites are showing the positive IFA pattern while other smaller E. acervulina sporozoites (arrows) are not labeled. X 2000. FIG. 6. General internal IFA pattern seen with an E. acervulina species-specific hybridoma antibody (designated 12-04) with the same mixed population of sporozoites seen in Figures 4 and 5. Note that only the E. acervulina sporozoites are demonstrating labeling while the E. tenella sporozoites (arrows) are not labeled. X 2000.
REFERENCES Bontemps, M., and P. Yvore, 1974. Technique de purification de suspensions de sporozoites d' Eimeria sur colonee de fibres synthetiques. Ann. Rech. Vet. 5:109-113. Danforth, H. D., 1982. Development of hybridomaproduced antibodies directed against Eimeria tenella and E. mitis. J.Parasitol. 68:392-397. Doran, D. J., and J. M. Vetterling, 1967. Cultivation of the turkey coccidium Eimeria meleagrimitis Tyzzer, 1929, in mammalian kidney cell cultures. Proc. Helminthol. Soc. Washington 34:59—65. Kennett, R. H., K. A. Denis, A. S. Tung, and N. R. Klinman, 1978. Hybrid plasmacytoma production: Fusions with adult spleen cells, monoclonal spleen fragments, neonatal spleen cells and human spleen cells. Current Topics in Microbiology and Immunology. R. Melchers, M. Potter, and N. L. Warner, ed. Springer-Verlag. New York, NY. Levine, P. P., 1938. Eimeria bagani n.sp (Protozoa: Eimeriidae) a new coccidium of the chicken. Cornell Vet. 28:263-266. Levine, P. P., 1942. A new coccidium pathogenic for chickens. Eimeria brunetti n.sp (Protozoa:
Eimeriidae). Cornell Vet. 32:430-439. Long, P. L., B. J. Millard, L. P. Joyner, and C. C. Norton, 1976. A guide to laboratory techniques used in the study and diagnosis of avian coccidiosis. Folia Vet. Lat. 6:201-217. Moore, E. N., and J. A. Brown, 1951. A new coccidium pathogenic for turkeys, Eimeria adenoeides n.sp (Protozoa: Eimeriidae). Cornell Vet. 41:124-135. Rose, M. E., 1973. Immunity. Pages 2 9 5 - 3 4 1 in The Coccidia: Eimeria, Isospora, Toxoplasma, and Related Genera. D. M. Hammond and P. L. Long, ed. University Park Press, Baltimore, MD. Rose, M. E., 1978. Immune responses of chickens to coccidia and coccidiosis. Pages 297—336 in Avian Coccidiosis. P. L. Long, K. N. Boorman, and B. M. Freeman, ed. Br. Poult. Sci. Ltd. Endinburgh. Rose, M. E., 1982. Host immune responses. Pages 3 2 9 - 3 7 1 in The Biology of the Coccidia. P. L. Long, ed. University Park Press, Baltimore, MD. Taylor, D. W., J. K. Kim, P. A. Munoz, C. B. Evans, and R. Asofsky, 1981. Monoclonal antibodies to stage-specific, species-specific, and cross-reactive antigens of the rodent malarial parasite Plasmodium yoelii. Infecti. Immun. 32:563—570.
chicken sera and t h e m o n o c l o n a l antibodies o n seven species of chicken and t w o species of t u r k e y coccidia. MATERIALS AND METHODS Immune Chicken Serum (ICS) Production. White Leghorn chickens raised coccidia-free were immunized against t h e following species and strains of coccidia b y giving t h e m six oral inoculations of 2 0 , 0 0 0 o o c y s t s during a 3-week interval: E. acervulina (API No. 12), E. maxima (API No. 4 1 ) , E. mitis (API No. 50), E. brunetti (API N o . 32), E. necatrix (API N o . 4 8 and 52), and E. tenella (API No. 10 and 2 4 ) . T h e birds were bled 7 days after t h e final inoculation, and t h e ICS were recovered and stored in 2-ml aliquots at —80 C until used. Preparation of Sporozoites. Sporozoites of t h e chicken coccidial species E. acervulina (API N o . 12), E. mitis (API N o . 50), E. mivati (API N o . 6), E. maxima (API N o . 4 1 ) , and E. tenella (API N o . 2 4 ) , and t h e t u r k e y species E. adenoeides (API N o . 20) and E. meleagrimitis (API N o . 4) were excysted and cleaned (Doran and Vetterling, 1 9 6 7 , B o n t e m p s and Yvore, 1 9 7 4 ) . These parasites were used t o i m m u n i z e mice for development of h y b r i d o m a cell lines and were also air dried o n i m m u n o f l u o r e s c e n t a n t i b o d y slides.
2145
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Production of Hybridoma Antibodies. The procedures for production of hybridoma antibodies (Ab) against Eimeria species have been previously described (Danforth, 1982). Briefly, Balb/CByJ mice (Jackson Laboratory, Bar Harbor, Maine) were immunized via tail vein injection with sporozoites of the species of chicken and turkey coccidia previously listed. The mice were given three injections of 100,000 viable sporozoites at 2-week intervals and a fourth injection one week later. Spleens were removed from the mice 3 days after the last injection, forced through a 60-mesh screen, mixed at a 10:1 ratio with P3-X63-Ag8 mouse myeloma cells, and fused in the presence of polyethylene glycol (Baker Co., Phillipsburg, NJ) as described by Kennett et al. (1978). The hybridoma cells produced by the fusion procedure were grown in Dulbecco's medium containing hypoxanthine, aminopterin and thymidine in 96-well, flat bottom microtiter plates (Falcon) (Danforth, 1982). Cell culture supernatants from wells with cell growth were screened for Ab production by the indirect immunofluorescent antibody (IFA) test using air-dried sporozoites of the same coccidial species against which the mice were immunized. Fifty out of 600 hybridoma cell lines that produced Ab against various species of sporozoites were then cloned by the limiting dilution technique previously described (Danforth, 1982). Based on the type of immunofluorescent pattern produced on or in air-dried sporozoites, 24 clones (Tables 2 and 3) were chosen to determine species-specificity or crossreactivity with the species and strains of Eimeria previously listed. Staining by Immunofluorescent Antibody. Air-dried sporozoites were first exposed to ICS or cell culture supernatant containing the hybridoma Ab for 30 min at 37 C. The slides were washed for 10 min in phosphate-buffered saline (PBS) at pH 7.3 and then treated for 30 min at 37 C with a pretitrated dilution of fluorescein-conjugated rabbit anti-mouse IgG (Miles Lab, Elkhart, IN). The slides were then washed for 10 min in fresh PBS, mounted with coverslips in the presence of glycerol, and viewed with a Zeiss ultraviolet epifluorescence microscope.
RESULTS
The ICS produced against individual species of avian coccidia were, in most cases, found to
crossreact with the sporozoites of eight chicken and two turkey species of coccidia (Table 1). The exceptions were the E. acervulina (API No. 12) ICS, which did not react with either E. adenoeides or E. meleagrimitis, and the E. maxima (API No. 41) and E. tenella (API No. 24) ICS that did not react with E. mivati sporozoites. Seven of the 24 monoclonal hybridoma antibodies were found to be species-specific (Table 2) and 17 were crossreactive (Table 3). Both the species-specific and the crossreactive antibodies demonstrated four different IFA patterns of tip, surface, refractile body, and internal (Tables 1 and 2; Figs. 1 to 4). In addition, one species-specific antibody directed against E. tenella had an internal anterior V4 IFA pattern, which was not seen with any of the crossreactive monoclonal antibodies (Table 2), and one crossreactive clone produced an IFA pattern that labeled both surface pellicle and internal area of the parasite, which was not observed with any of the species-specific Ab (Table 3). The hybridoma antibodies that labeled the refractile bodies of the sporozoites were usually crossreactive with all species of coccidia tested (Table 3 and Fig. 3). However, one monoclonal Ab directed against E. adenoeides (designated 91-C7) labeled only the refractile body of this species (Table 2). Two other monoclonal Ab, designated 12-27 and 95-G10, that labeled the surface and surface-interior, respectively, also crossreacted with all species of coccidia tested (Table 3). Some of the crossreactive hybridoma Ab demonstrated one pattern with the homologous sporozoites and a still different pattern with sporozoites of other species (Tables 3 and 4). These variations were seen for all IFA patterns except the refractile body, which was area specific, regardless of species. In some cases, there was much variation in IFA patterns. For example, Ab from the 12-27 clone labeled the surface of the homologous E. acervulina clone sporozoites but gave either a tip IFA pattern with E. adenoeides or an internal IFA pattern with E. meleagrimitis (Table 4). Similarly, clone 95—G7, that gave a tip IFA pattern against E. adenoeides, gave a surface-internal pattern with E. mivati and E. maxima sporozoites (Table 4). Other monoclonal Ab that showed either an internal IFA pattern (94-G2) or combination surface-internal pattern (95G10) on the E. adenoeides sporozoites that
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they were directed against, labeled other areas of the heterologous, crossreactive sporozoites (Table 4). Antibody produced against the E. adenoeides turkey species demonstrated greater variation in IFA patterns than Ab produced against any of the chicken species (Table 4). By use of crossreactive and species-specific monoclonal Ab, it is possible to identify and speciate coccidial parasites in mixed infections (Figs. 4-6). A mixed population of coccidia is easily recognized by use of the 12-27 crossreactive Ab (Table 3; Fig. 4), and then speciation of the mixed population, in this example E. tenella and E. acervulina, can be accomplished with the species-specific F8-3B7 and 12-04 hybridoma Ab (Figs. 5 and 6). In this way, it is also possible for laboratory strains maintained at the Poultry Parasitic Diseases Laboratory to be periodically checked for purity.
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This investigation has shown that it is possible to produce both crossreactive and species-specific hybridoma antibodies against various species of avian coccidia. This is similar to hybridoma antibodies directed against other protozoan parasites such as Plasmodium yoelli (Taylor et al, 1981) in which species-specificity and crossreactivity between red blood cell stages was also observed with various hybridoma cell lines. Unlike the sera from birds immunized against a single species of coccidia that crossreacts with other species, some hybridoma Ab are species-specific and can be used to identify coccidia species in mixed infections.
TABLE 2. Species-specific hybridoma
antibodies
Cloned cell line
Speciesspecificity
Immunofluorescent pattern 1
F8-3B7 B6-1H4 12-04 12-23 05-2F11 76-G6 91-C7
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1 1 = Internal; T = tip; S = surface; RB = refractile body immunofluorescent antibody patterns.
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These results also indicate that identification of the species of coccidia present in field litter samples can be quickly made without the use of bird immunization (Levine, 1938, 1942; Moore and Brown, 1951; Long, et al, 1976). Whether strain variations would occur with hybridoma Ab such as was seen with bird immunity to E. acervulina and E. maxima (Rose, 1978; 1982) will require further investigation. However, it has been previously reported that there is no apparent strain specificity seen with speciesspecific hybridoma Ab directed E. tenella sporozoites (Danforth, 1982). Eimeria species elicit specific immunity (Rose, 1973; 1978). Accordingly, there must be some antigen(s) or antigenic determinant(s) specific to single species of coccidia for such immunity to be elicited in birds. Thus, it is to be expected that some of the hybridoma Ab would react with only the species of coccidial sporozoite against which they were raised. Shared antigen or antigenic determinants between coccidial species have also been demonstrated (Rose, 1982). Hence, it was not surprising that Ab directed against crossreactive antigenic determinants were produced in the hybridoma model. It is evident that during the evolutionary development of coccidial parasites, some common antigens were retained or conserved, while others unique to the specific species of coccidia were developed. It is not known why there were different IFA patterns seen on sporozoites exposed to some of the crossreactive hybridoma Ab. The antigenic determinants recognized by the Ab are apparently located in different areas of these sporozoites. Whether this crossreactivity indicates the presence of homologous molecules must await further biochemical analysis. Efforts are now underway to produce hybridoma antibodies directed against the remaining pathogenic species of chicken and turkey coccidia. Preliminary evidence suggests that a diagnostic procedure utilizing an enzyme-linked immunoadsorbent assay (ELISA) could then be used for the diagnosis of species of coccidia in commercial poultry houses.
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The authors wish to thank Michael Ruff, Gary Wilkins, and Jeff Fagen for providing the immune chicken serum, and Maryalice Miller, Pauldo Smith, and Nancy Koles for maintenance of the hybridoma cell lines.
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DENFORTH AND AUGUSTINE
RESPONSES TO HYBRIDOMA AND COCCIDIAL ANTIGENS
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FIG. 1. Specific tip immunofluorescent antibody (IFA) pattern seen with E. adenoeides sporozoites exposed to a hybridoma antibody (designated 95-G9) that was originally raised against this same species of parasite. X 2000. FIG. 2. Surface IFA pattern seen on E. maxima sporozoites exposed to a hybridoma antibody (designated 12-27), which showed a surface-internal IFA pattern with the E. acervulina sporozoites against which it was originally raised. X 2000. FIG. 3. Refractile body IFA pattern seen with E. adenoeides sporozoites exposed to a hybridoma antibody (designated 91C7) that was originally raised against this species of parasites. Although this particular refractile body hybridoma was species-specific, the other crossreactive refractile body hybridomas demonstrated the same type of IFA pattern. FIG. 4. Surface-internal IFA pattern demonstrated by the 12-27 crossreactive hybridoma when it was exposed to a mixed population of parasites (E. tenella and E. acervulina). X 2000. FIG. 5. General internal IFA pattern seen with an E. tenella species-specific hybridoma antibody (designated F8-3B7) with the same mixed population of sporozoites used in Figure 4. Note that only the E. tenella sporozoites are showing the positive IFA pattern while other smaller E. acervulina sporozoites (arrows) are not labeled. X 2000. FIG. 6. General internal IFA pattern seen with an E. acervulina species-specific hybridoma antibody (designated 12-04) with the same mixed population of sporozoites seen in Figures 4 and 5. Note that only the E. acervulina sporozoites are demonstrating labeling while the E. tenella sporozoites (arrows) are not labeled. X 2000.
REFERENCES Bontemps, M., and P. Yvore, 1974. Technique de purification de suspensions de sporozoites d' Eimeria sur colonee de fibres synthetiques. Ann. Rech. Vet. 5:109-113. Danforth, H. D., 1982. Development of hybridomaproduced antibodies directed against Eimeria tenella and E. mitis. J.Parasitol. 68:392-397. Doran, D. J., and J. M. Vetterling, 1967. Cultivation of the turkey coccidium Eimeria meleagrimitis Tyzzer, 1929, in mammalian kidney cell cultures. Proc. Helminthol. Soc. Washington 34:59—65. Kennett, R. H., K. A. Denis, A. S. Tung, and N. R. Klinman, 1978. Hybrid plasmacytoma production: Fusions with adult spleen cells, monoclonal spleen fragments, neonatal spleen cells and human spleen cells. Current Topics in Microbiology and Immunology. R. Melchers, M. Potter, and N. L. Warner, ed. Springer-Verlag. New York, NY. Levine, P. P., 1938. Eimeria bagani n.sp (Protozoa: Eimeriidae) a new coccidium of the chicken. Cornell Vet. 28:263-266. Levine, P. P., 1942. A new coccidium pathogenic for chickens. Eimeria brunetti n.sp (Protozoa:
Eimeriidae). Cornell Vet. 32:430-439. Long, P. L., B. J. Millard, L. P. Joyner, and C. C. Norton, 1976. A guide to laboratory techniques used in the study and diagnosis of avian coccidiosis. Folia Vet. Lat. 6:201-217. Moore, E. N., and J. A. Brown, 1951. A new coccidium pathogenic for turkeys, Eimeria adenoeides n.sp (Protozoa: Eimeriidae). Cornell Vet. 41:124-135. Rose, M. E., 1973. Immunity. Pages 2 9 5 - 3 4 1 in The Coccidia: Eimeria, Isospora, Toxoplasma, and Related Genera. D. M. Hammond and P. L. Long, ed. University Park Press, Baltimore, MD. Rose, M. E., 1978. Immune responses of chickens to coccidia and coccidiosis. Pages 297—336 in Avian Coccidiosis. P. L. Long, K. N. Boorman, and B. M. Freeman, ed. Br. Poult. Sci. Ltd. Endinburgh. Rose, M. E., 1982. Host immune responses. Pages 3 2 9 - 3 7 1 in The Biology of the Coccidia. P. L. Long, ed. University Park Press, Baltimore, MD. Taylor, D. W., J. K. Kim, P. A. Munoz, C. B. Evans, and R. Asofsky, 1981. Monoclonal antibodies to stage-specific, species-specific, and cross-reactive antigens of the rodent malarial parasite Plasmodium yoelii. Infecti. Immun. 32:563—570.