Characterization of Immune Response to Eimeria tenella Antigens in a ...

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Oct 18, 1996 - Immunosuppression in protozoal infections, p. 431–462. In M. Levandowsky and S. H. Hunter (ed.), Biochem- istry and physiology of protozoa.

INFECTION AND IMMUNITY, Apr. 1997, p. 1204–1210 0019-9567/97/$04.0010 Copyright q 1997, American Society for Microbiology

Vol. 65, No. 4

Characterization of Immune Response to Eimeria tenella Antigens in a Natural Immunity Model with Hosts Which Differ Serologically at the B Locus of the Major Histocompatibility Complex DAVID A. BRAKE,* CAROLYN H. FEDOR,† BRENDA W. WERNER, TIMOTHY J. MILLER,‡ ROBERT L. TAYLOR, JR.,§ AND ROBERT A. CLAREi Animal Health Biological Discovery, Pfizer Central Research Division, Pfizer, Inc., Groton, Connecticut 06340 Received 30 August 1996/Returned for modification 18 October 1996/Accepted 7 January 1997

A model to simulate natural immunity to Eimeria tenella was developed in three chicken lines which differ at the B locus of the major histocompatibility complex. Homozygous, 1-day-old chicks of the B19B19, B24B24, or B30B30 genotype were trickle immunized by being orally fed a small infectious dose of E. tenella oocysts for 5 consecutive days. These naturally exposed birds were then challenged at different times between 5 and 24 days after the final dose, and the level of protection was assessed 6 days after challenge, using body weight gain and intestinal lesion scores. The duration of immunity in naturally exposed birds differed among the major histocompatibility complex lines. Trickle immunization of the B19B19 haplotype afforded the longest and strongest level of protection compared to the other two haplotypes tested. In addition, in vitro splenic and peripheral blood lymphocyte proliferative responses in trickle-immunized birds were measured against sporozoite, merozoite, and tissue culture-derived E. tenella parasite antigens isolated from the recently described SB-CEV-1/F7 established cell line. The lymphocytes obtained from B19B19 birds trickle immunized responded in vitro to the E. tenella-infected SB-CEV-1/F7 tissue culture-derived parasite antigen. Furthermore, antigen-specific immune responses appeared earlier in immune, challenged B19B19 birds than in their naive, challenged counterparts. The development of a model simulating natural immunization will serve as a foundation to further characterize both humoral and cell-mediated responses to E. tenella tissue culture-derived parasite antigens and to better understand host protective immune responses to avian coccidiosis. tion to provide a clearer understanding of the host-parasite interaction. Trickle immunization, or exposure of chickens to small graded doses of infectious oocysts over a relatively short time span, will induce a state of immunity against homologous challenge (21). This immunity has not been adequately studied to better define the host protective immune mechanisms to Eimeria infection. Rather, the vast majority of immune responses to avian Eimeria spp. have been measured in birds which have previously undergone unnatural multiple, high-level rounds of reinfection (10, 15). Also, from a vaccine standpoint, the more natural trickle immunization model has not been applied to the eludication of specific parasite gene products responsible for the induction of host protection. Based on the concept of trickle immunization, live virulent and avirulent (precocious line) oocyst vaccines have been proven efficacious and are commercially available (51). However, perceived problems in controlled administration, pathogenicity, flock management, and other issues warrant the identification of protective parasite antigens which can be incorporated into a safer form of vaccination. Several reports provide data which together suggest that protective immunity against clinical coccidiosis is predominantly cell mediated (for a recent review, see reference 28) and directed toward Eimeria gene products expressed during the intracellular, asexual developmental stage (15, 24, 31, 32, 38, 44). Our laboratory has recently described a new, continuous cell line which supports high levels of Eimeria tenella intracellular development and merogony (11). The present study was designed in part to unify the phenomenon of trickle immuni-

At present, the most economically important parasitic disease complex in poultry management is avian coccidiosis. Clinical disease, manifested by severe enteritis with associated morbidity and mortality, is caused by several species of the genus Eimeria, an obligate intracellular protozoan that invades the mucosa and lamina propria portions of the intestinal tract (29). Whereas current control strategies based on prophylactic chemotherapy are effective in preventing severe weight loss and growth depression, the cost of anticoccidial drug development and treatment coupled with the rapid emergence of drugresistant parasites has prompted the search for more costeffective and safer alternatives for coccidiosis control (10, 28). For example, over the past several years the cloning, characterization, and expression of numerous Eimeria spp. sporozoite, merozoite, and gametocyte genes have been described (5, 18, 34, 37, 57). Although considerable and significant genetic information has been generated from the molecular cloning approach, a single subunit coccidiosis vaccine has yet to be delivered successfully to the poultry marketplace. Perhaps further efforts should be directed at obtaining biological informa-

* Corresponding author. Mailing address: Animal Health Biological Discovery, Pfizer Central Research Division, Pfizer Inc., Eastern Point Road, Groton, CT 06340. Phone: (860) 441-8230. Fax: (860) 441-8739. E-mail: [email protected] † Present address: SmithKline Beecham, King of Prussia, PA 19406. ‡ Present address: BioSense Inc., Lincoln, NE 65806. § Present address: Department of Animal and Nutritional Sciences, University of New Hampshire, Durham, NH 03824. i Present address: SmithKline Beecham, Collegeville, PA 19426. 1204

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zation with the ability to produce high amounts of E. tenella parasite antigens in vitro, as an initial strategy to characterize E. tenella tissue culture-derived parasite antigens which may be involved in the induction of host protection. Since the chicken major histocompatibility (MHC) (50) or B complex (4) has been implicated in conferring immunocompetence against E. tenella (6–8, 36), this study also used three MHC bird lines which differ serologically at the B locus. This report describes the results and important features of natural immune models established in different, homozygous B genotype chicken lines and also demonstrates that immune lymphocytes obtained from these naturally exposed birds proliferate in response to parasite antigens obtained from E. tenella-infected tissue culture supernatants. MATERIALS AND METHODS Parasite source and preparation. E. tenella (strain LS 65) was used throughout this study. Oocysts were produced and maintained by routine passage in 3-weekold Peterson Arbor Acre broilers, and the inoculum was prepared as described previously (13), with the following exceptions. Oocysts were broken on ice for 3 min by using a bead beater (Biospec Products, Bartlesville, Okla.), and sporocysts were purified by using 0.75 M sucrose instead of 1 M sucrose. Preparation of sporozoite and merozoite antigens. Sporozoites and in vitroproduced merozoites (see below) were resuspended in 13 Dulbecco’s phosphate-buffered saline (PBS) (pH 7.2) containing 0.5 mM phenylmethylsulfonyl fluoride (Calbiochem-Behring, La Jolla, Calif.), freeze-thawed three times on dry ice, and sonicated (model W-380; Heat Systems Ultrasonics, Farmingdale, N.Y.) on ice, using a 1-s pulse and 80% duty cycle. After five cycles, each 1 min long, samples were transferred to microcentrifuge tubes and centrifuged at 10,000 3 g for 10 min at 48C. Soluble material above the pellet was collected, and the protein concentration was determined by the method of Bradford (1). Sonicated parasite preparations were adjusted to 1 mg/ml in serum-free medium 199 (BRL Life Technologies, Grand Island, N.Y.), aliquoted, and stored at 2208C until use. Preparation of tissue culture-derived parasite antigens. E. tenella parasite antigens were produced in the SB-CEV-1/F7 cell line (ATCC CRL10495). Tissue culture vessels (150 cm2) were seeded with SB-CEV-1/F7 cells at a density of 105/ml suspended in 30 ml of medium 199 with 5% fetal bovine serum and incubated at 40.58C and 5% CO2. After an overnight incubation to obtain confluency, medium was aspirated and sporozoites were added at a density of 106 sporozoites/ml in 30 ml of medium 199. Between 2 and 24 (24 h), 24 and 48 (48 h), and 48 and 72 (72 h) h postinfection, conditioned medium was removed from infected cultures, pooled, and centrifuged at 800 3 g for 15 min at 48C to remove cell debris and any intact parasite forms. Pellets from the 72-h harvest contained in vitro-produced merozoites and were used for the preparation of sonicated merozoite antigen as described above. Tissue culture supernatant samples from each time point were quantitated for parasite-specific protein by using a direct sporozoite enzyme-linked immunosorbent assay (ELISA). Briefly, polyclonal antisporozoite serum was obtained from rabbits injected twice with sporozoites emulsified in Freund’s complete adjuvant at weekly intervals. Tissue culturederived parasite antigens (100 ng/well) and purified sporozoites (10 ng/well) were absorbed to ELISA plates (Nunc, Inc., Naperville, Ill.) overnight at 48C. After washing with phosphate-buffered saline, the rabbit antisporozoite serum (1: 20,000) was added and incubated at 378C for 1 h. Following three PBS washes, biotin-labeled goat anti-rabbit antibody (Kirkegaard & Perry, Gaithersburg, Md.) was added and incubated 1 h at 378C. Following three PBS washes, peroxidase-labeled streptavidin (Kirkegaard & Perry) diluted in 2% skim milk was added and incubated for 1 h at 378C, followed by three PBS washes and the addition of 3,39,5,59-tetramethylbenzidine-peroxidase in hydrogen peroxide. The reaction was stopped with 1 M hydrochloric acid, and plates were read at 450 nm. Cell-free supernatants were aliquoted and frozen at 2208C until use. Samples were thawed immediately prior to use for in vitro assays. Chickens. One-day-old B19B19 White Leghorn or B24B24 and B30B30 New Hampshire chickens (New Hampshire Poultry Research Center, University of New Hampshire, Durham) hatched from the eggs of hens maintained under coccidium-free conditions were vaccinated against Marek’s disease and wingbanded. Chicks were housed in wire-floor battery cages in an American Association for Laboratory Animal Care facility, and commercial starter-growing ration (Purina Co., Indianapolis, Ind.) and water were provided for ad libitum consumption. Trickle immunizations and parasite challenge. Birds were orally inoculated with either distilled water or 500 sporulated E. tenella oocysts in 1.0 ml of distilled water for 5 consecutive days. Coccidium-free, naive birds were housed in a separate room. At various ages, birds were weighed and orally challenged with homologous oocysts. Clinical criteria. Chickens used in all experiments were handled according to the guidelines of the site Institutional Use and Care Committee. Clinical disease was monitored 6 days postchallenge, using the criteria of body weight gain (days

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0 to 6 postchallenge) and cecal lesion scores. Prior to harvesting of spleens or lesion scoring, chickens were sacrificed by cervical dislocation. Lesions were scored on a scale of 0 (no damage to cecal mucosal tissue) to 4 (maximum damage to cecal mucosal tissue) as previously described (19). Cell isolation and in vitro proliferation assays. Blood was collected from the brachial vein into heparinized Vacutainer tubes (Becton-Dickinson, Mountainview, Calif.). Blood samples were diluted 1:3 in Ca21- and Mg21-free Hanks’ balanced salt solution containing 25 mM HEPES, pH 7.5 (CHBSS) (Bio-Rad Laboratories, Richmond, Calif.), and washed two times at 150 3 g for 10 min at room temperature. Samples were diluted to twice the original volume in CHBSS, and 3 ml of the cell suspensions was layered over an equal volume of Histopaque 1077 (Sigma Chemical Company, St. Louis, Mo.). Samples were centrifuged at 400 3 g for 15 min at room temperature, and cells from the interface were collected. Spleens were removed and placed in ice-cold sterile CHBSS. Spleen tissue was disrupted in CHBSS by using a sterile scalpel and a 14-gauge cannula. Cell suspensions were centrifuged at 50 3 g for 10 min at room temperature. Supernatants were removed and centrifuged at 150 3 g for 10 min at room temperature. Cell pellets were resuspended in CHBSS, and 3-ml suspensions were layered over Histopaque and centrifuged as described above. Cell interfaces from blood and spleen suspensions were collected, diluted in CHBSS, and washed three times at 150 3 g for 10 min at room temperature. Viable cells were enumerated by using trypan blue and a hemacytometer. For in vitro proliferation assays, peripheral blood lymphocytes (PBL) and spleen cells were adjusted to a density of 107 cells/ml in complete serum-free Leibovitz’s modified Hahn’s media (equal parts McCoy’s 5A and Leibovitz’s media, 5 3 1025 M 2-mercaptoethanol, 5 mg of insulin per ml, 2 mM L-glutamine, 100 U of penicillin and streptomycin per ml, 0.25 mg of amphotericin B per ml, 2% tryptose phosphate, 1 mM sodium pyruvate) (Gibco Life Technologies). Sonicated sporozoite and merozoite extracellular parasite antigens (5 mg/ ml), serum-free infected SB-CEV-1/F7 tissue culture supernatants (see text for concentrations), concanavalin A (ConA; 10 mg/ml; Sigma), or serum-free medium 199 was thawed at 378C and added (0.1 ml/well) to 96-well round-bottom microtiter plates (Linbro, Flow Laboratories). For PBL proliferation experiments, autologous erythrocytes (107/ml) were added (0.05 ml/well). The PBL or splenic lymphocytes were then added (0.05 ml/well) in quadruplicate. For mitogen and antigen proliferation assays, cultures were incubated at 408C under 5% CO2 for 72 and 96 h, respectively, and pulsed with [3H]thymidine, 1 mCi/well (5.0 mCi/mmol, specific activity; Amersham, Arlington Heights, Ill.), during the final 18 h of culture. Cells were harvested onto glass fiber mats by using an automated, programmable 96-well harvester (MACH III; TomTech, Orange, Conn.), and radioactivity was determined in a direct beta counter (Matrix 96; Packard Instrument Co., Sterling, Va.). For the antigen proliferation assays, results are expressed as a stimulation index (SI) previously described (40) in which SI equals [(mean cpm test lymphocytes plus antigen)/(mean cpm test lymphocytes in medium alone)] divided by [(mean cpm control lymphocytes plus antigen)/(mean cpm control lymphocytes in medium alone)]. The SI values for the control lymphocyte cultures were similar throughout the experiments. Statistics. The weight gain and lesion score data were analyzed by the general linear models procedure (48). Least-squares statistical comparisons for weight gain and lesion score were made between the naive control groups and trickleimmunized groups after both groups were challenged with E. tenella oocysts. P # 0.05 values are considered significant.

RESULTS Protection against disease in trickle-immunized B19B19 birds. Trickle-immunized B19B19 and age-matched naive birds were challenged orally with 35,000 E. tenella oocysts when they were 10, 16, 22, 25, or 29 days of age. Birds previously vaccinated by trickle immunization and challenged 5, 11, 17, or 20 days following the last oocyst exposure were significantly protected against weight loss compared to naive, challenged controls (Table 1). However, vaccinated birds challenged 24 days following the last day of trickle feeding were not protected against weight loss. The lesion scores obtained at the various challenge time points paralleled the weight gain results in that significant protection was observed when immune birds were challenged up to 20 days following the last parasite exposure (Table 1). These observations indicate that the duration of immunity is between 3 and 4 weeks in 1-day-old B19B19 birds naturally exposed to 500 oocysts per day for 5 consecutive days. Protection against disease in trickle-immunized B24B24 birds. Trickle-immunized day-old B24B24 and age-matched naive birds were challenged orally with E. tenella oocysts when they were either 16, 18, or 21 days of age. Clinical results

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TABLE 1. Mean weight gains and lesion scores following challenge with E. tenella of B19B19 immunized and naive birds

TABLE 3. Mean weight gains and lesion scores following challenge with E. tenella in B30B30 immunized and naive birds

Treatmenta

Challenge age (days)b

n

Wt gain (g)c

Lesion scorec

Treatmenta

Challenge age (days)b

Challenge dose (104)

n

Wt gain (g)c

Lesion scorec

Immunized Naive Immunized Naive Immunized Naive Immunized Naive Immunized Naive

10 10 16 16 22 22 25 25 29 29

10 7 8 7 5 5 10 10 5 5

51d 29 73e 66 78d 34 88e 67 110 86

0.4d 3.2 0.9d 3.0 0.9d 3.4 0.5f 2.0 1.0 2.4

Immunized Naive Immunized Naive Immunized Naive Immunized Naive

16 16 18 18 21 21 22 22

3.5 3.5 3.5 3.5 1.0 1.0 1.0 1.0

8 8 8 7 5 4 10 5

86d 71 72d 45 82d 59 73e 28

1.3e 2.9 3.1 2.9 2.4 3.0 1.6d 3.4

a One-day-old birds were immunized by oral inoculation with 500 oocysts for 5 consecutive days. Naive birds received distilled water orally. b Birds were challenged by oral gavage with 3.5 3 104 E. tenella oocysts. c Determined 6 days after challenge. d P # 0.005 when comparing immunized birds to naive controls after challenge. e P # 0.05 when comparing immunized birds to naive controls after challenge. f P # 0.01 when comparing immunized birds to naive controls after challenge.

(Table 2) indicate that trickle-vaccinated birds were significantly protected against both weight loss and lesions when challenged at 16 or 18 days of age. In contrast, 21-day-old vaccinated birds were susceptible to E. tenella challenge with either 10,000 or 35,000 oocysts. Although the 10,000-oocyst challenge dose resulted in a marked reduction in the severity of lesions compared to the 35,000-oocyst challenge dose, lesion scores in immunized birds did not differ significantly from those for naive controls. These results indicate that the duration of immunity is approximately 2 weeks in 1-day-old B24B24 birds, using the described trickle vaccination regimen. Protection against disease in trickle-immunized B30B30 birds. Trickle-immunized day-old B30B30 and age-matched naive birds were challenged with 35,000 oocysts at 16 or 18 days of age. Other groups were challenged with 10,000 oocysts at 21 or 22 days of age. Clinical coccidiosis was observed in naive birds challenged at either 16 or 18 days of age (Table 3). Conversely, 16- and 18-day-old vaccinated birds were significantly protected against weight loss after challenge. In addition, although the 16-day-old trickle-immunized, challenged birds were significantly protected against lesions, a group challenged 2 days later developed a high number of lesions. A

a One-day-old birds were immunized by oral vaccination with 500 oocysts for 5 consecutive days. Naive birds received distilled water orally. b Birds were challenged by oral gavage with the indicated number of E. tenella oocysts. c Determined 6 days after challenge. d P # 0.05 when comparing immunized birds to naive controls after challenge. e P # 0.01 when comparing immunized birds to naive controls after challenge.

similar trend was also noted in 21-day-old trickle-vaccinated birds challenged with 10,000 oocysts; i.e., significant protection was observed against weight loss but not against lesion score. Interestingly, birds challenged only 24 h later (22 days old) were protected against both weight loss and lesions. These data indicate that in trickle-immunized day-old B30B30 birds, the duration of immunity against E. tenella challenge is at least 3 weeks. Comparison of disease protection and mitogen responses in naive and immune, challenged birds. To compare disease protection with host cellular immune responses, day-old B19B19 trickle-immunized and naive birds were challenged with E. tenella oocysts at 10 days of age, and weight gain, lesion scores, and ConA responses were evaluated 6 days later (Table 4). Trickle-immunized birds were significantly resistant to acute challenge 5 days after their last oocyst trickle dose. The PBL obtained from these immune birds at day 6 postchallenge showed strong proliferative responses to the T-cell mitogen ConA. A similar pattern was observed over a range of ConA concentrations, and immune PBL response levels were similar to levels obtained in age-matched naive, unchallenged birds (data not shown). In striking contrast, PBL from naive, challenged controls exhibited a weak ConA response. Surprisingly, splenic lymphocytes from these same animals showed mitogenic responses comparable to levels obtained in immune, challenged birds. Based on these observations, there appeared

TABLE 2. Mean weight gains and lesion scores following challenge with E. tenella in B24B24 immunized and naive birds Treatmenta

Challenge age (days)b

Challenge dose (104)

n

Wt gain (g)c

Lesion scorec

Immunized Naive Immunized Naive Immunized Naive Immunized Naive

16 16 18 18 21 21 21 21

3.5 3.5 3.5 3.5 1.0 1.0 3.5 3.5

5 8 8 8 5 5 5 5

77d 68 86d 66 94 95 90 83

1.8e 3.0 2.3d 3.1 0.8 1.2 2.4 3.0

a One-day-old birds were immunized by oral vaccination with 500 oocysts for 5 consecutive days. Naive birds received distilled water orally. b Birds were challenged by oral gavage with the indicated number of E. tenella oocysts. c Determined 6 days after challenge. d P # 0.05 when comparing immunized birds to naive controls after challenge. e P # 0.005 when comparing immunized birds to naive controls after challenge.

TABLE 4. Comparison of weight gains, lesion scores, and mitogenic responses following challenge with E. tenella in B19B19 immunized and naive birds Treatmentb

Immunized Naive

Wt gain (g)c d

40 28

Lesion scorec e

0.8 3.4

Mean ConA response (SI)a 6 SD PBL

Spleen lymphocyte

59.6 6 7.8 0.7 6 0.2

8.5 6 1.1 16.5 6 1.7

a Lymphocytes (5 3 105) were cocultured with 5 3 105 autologous erythrocytes and ConA (10 mg/ml). SI 5 (mean cpm 1 ConA)/(mean cpm 1 medium alone). b One-day-old birds (n 5 8) were immunized by oral vaccination with 500 oocysts for 5 consecutive days. Naive birds (n 5 8) received distilled water orally. Birds were challenged by oral gavage at 10 days of age with 3.5 3 104 E. tenella oocysts. c Determined 6 days after challenge. d P # 0.05 when comparing immunized birds to naive controls after challenge. e P # 0.005 when comparing immunized birds to naive controls after challenge.

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DISCUSSION Continuous low-level infection with oocysts, termed trickleimmunization, will induce solid protection against clinical coccidiosis (21, 22, 53). Although some permutations on this general phenomenon have been reported (12, 14, 35), this natural model has not been studied adequately with the objective of developing a more thorough understanding of the underlying host immune mechanisms associated with protection, particularly in young chickens. Historically, in vitro immune measurement studies using avian Eimeria spp. have commonly used FIG. 1. Proliferative responses of E. tenella-immune B19B19 splenic lymphocytes to extracellular and tissue culture-derived parasite antigens. Splenic lymphocytes isolated from trickle-immunized birds at 25 days of age were stimulated in the presence of uninfected tissue culture media (UN); tissue culture supernatants harvested 24 h (24), 48 h (48), or 72 h (72) after infection with E. tenella; E. tenella sporozoites (spz), or merozoites (mrz). The parasite-specific protein concentration for each antigen was 0, 0.5, 0.7, 4.2, 2.5, or 2.5 mg/ml, respectively. Results are expressed as SIs (see Materials and Methods).

to be a relationship in B19B19 birds 6 days after challenge between disease protection and PBL responses to ConA. In vitro proliferation of lymphocytes from trickle-vaccinated and naive birds to various parasite antigens. To determine if SB-CEV-1/F7-produced E. tenella parasite antigens were capable of stimulating immune lymphocytes obtained from naturally infected birds, B19B19 splenic lymphocyte proliferative responses were tested in cultures containing either extracellular (sporozoite or merozoite) antigens or supernatants derived from infected tissue cultures. As expected, immune splenic lymphocytes cocultured with either sporozoite or merozoite antigen showed good stimulatory responses (Fig. 1). In addition, supernatants derived from infected tissue cultures harvested at either 24, 48, or 72 h postinfection induced higher stimulation indices compared to the uninfected, SB-CEV-1/F7 tissue culture supernatant control. However, due to the differences in parasite-specific protein concentrations between the various parasite antigen samples, it is difficult to determine which parasite antigen fraction possessed the best overall stimulatory activity. These data show that immune lymphocytes obtained from day-old birds exposed to E. tenella will proliferate in vitro when stimulated with parasite antigens derived from the tissue culture supernatant of an E. tenella-infected cell line. The SB-CEV-1/F7 cell line appears to support the in vitro expression and synthesis of E. tenella parasite gene products which can be recognized by lymphocytes from immune birds. Postchallenge response to tissue culture-derived parasite supernatants in naive and trickle-immunized birds. Naive and trickle-immunized B19B19 birds were challenged at 10 days of age with 35,000 E. tenella oocysts. PBL responses to infected tissue culture supernatants (Fig. 2) or sporozoites and merozoites (Fig. 3) were assessed on 4 consecutive days postchallenge. Overall, PBL from trickle-immunized, challenged birds cocultured with either sporozoite or merozoite antigens yielded the highest SIs. Interestingly, the highest PBL responses in trickle-immunized, challenged birds to the sporozoite and merozoite antigen samples tested occurred at day 4 postchallenge. In contrast, PBL responses in naive, challenged birds against these same antigens were delayed by 48 h and were highest at day 6 postchallenge. At day 4 postchallenge, PBL from trickleimmunized birds incubated in the presence of 24-, 48-, and 72-h supernatants derived from infected tissue cultures possessed low but detectable stimulatory activity.

FIG. 2. Proliferative responses of naive and E. tenella-immune B19B19 PBL to tissue culture-derived parasite antigen. PBL obtained 3 to 6 days postchallenge were stimulated in the presence of autologous erythrocytes and E. tenellainfected SB-CEV-1/F7 tissue culture-derived supernatants collected from 0 to 24 h postinfection (A), 24 to 48 h postinfection (B), and 48 to 72 h postinfection (C). The amounts of parasite-specific protein per assay were 1.25, 1.10, and 9.10 mg/ml, respectively.

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FIG. 3. Proliferative responses of naive and E. tenella-immune B19B19 PBL to sporozoite (A) or merozoite (B) antigen. PBL obtained 3 to 6 days postchallenge were stimulated in the presence of autologous erythrocytes and E. tenella sporozoites (5 mg/ml) or merozoites (5 mg/ml).

either a single-dose or multiple massive-dose infections to establish a solid state of immunity in older birds (26). Because the immune repertoire in an older bird differs considerably from the developing immune repertoire in a young chick (49), attempts to generalize from such systems should be interpreted with caution. A number of published reports related to Eimeria and host immunity have been conducted by using murine models of disease, due primarily to the availability of a betterdefined and broader range of immunological reagents (33, 43, 45–47, 54, 56). Although several important findings have been made in this system, the application of murine coccidial disease models to the understanding of clinical coccidiosis in domestic birds should be carefully considered. Because avian host immune responses to Eimeria have been demonstrated to be mediated in part by the MHC (7, 8, 20, 27), this study was undertaken to examine the duration of immunity following trickle immunization of 1-day-old chicks from three lines which differ at the B complex as well as other genes. Results show that the duration of immunity, using the regimen of 500 oocysts daily from 1 to 5 days of age, ranged between 2 and 3 weeks and was dependent on the bird line used. These results support previous observations which showed that natural oocyst exposure in newly hatched chicks 1 day of age, the target age for vaccination, imparts solid immunity against virulent, homologous challenge (12, 35). The present study extends these results and demonstrates further that (i) the duration of trickle vaccination immunity may be associated with the B-haplotype allele and (ii) a comparable duration of immunity can be induced by using a shorter trickle immunization period (5 days) than previously reported (14 days). A comparison of mean lesion scores between the three trickle-immunized bird lines following challenge indicates that trickle immunization of the B19B19 haplotype affords the strongest degree of protection

INFECT. IMMUN.

compared to the B24B24 and B30B30 haplotypes. In addition, there appears to be a dichotomy between weight gain and lesion scores in some E. tenella-immune birds. For example, at certain time points, trickle-immunized B30B30 birds were resistant to challenge based on weight gain but were susceptible to challenge based on lesion scores (Table 3). This observation may be related to the bird genotype, challenge age, dose, background genes, or combinations of the above. Another plausible explanation is that the immune mechanisms responsible for protection against weight loss may be distinct from those pathways operative to reduce intestinal lesions in specific bird lines, an argument supported by data obtained in studies using X-irradiated oocysts (17). From our study, it appears to be important to use at least two different criteria when evaluating protection against E. tenella coccidiosis. To this end, measurement of cellular immune responses was subsequently conducted in the B19B19 bird line since the haplotype displayed the most consistent and solid protection in the trickle immunization challenge model. An in vitro immune assay which correlates the immune status of a bird to its level of resistance against clinical coccidiosis is not presently available. It is generally accepted that circulating antibody titers to asexual-stage (i.e., sporozoite and merozoite) antigens do not correlate with protection (39), although maternal antibody titers may play some role in protection against challenge (52, 58). Whereas PBL and splenic T cells isolated from Eimeria-immune birds have been clearly shown to possess proliferative responses to various native and recombinant parasite antigens (25, 26), similar-T cell populations obtained from acutely infected, nonimmune hosts respond equally well to some of these same antigens (30). In vitro PBL and splenic blastogenic responses to the extracellular parasite antigens (sporozoite and merozoite) do not correlate with protection against weight loss and lesions, although a correlation between proliferation and oocyst shedding has been reported (55). In an attempt to identify an in vitro assay associated with disease protection, lymphocyte mitogenic responses were compared between naive and immune, challenged birds. Results show that in naive, but not trickle-immunized, birds, the PBL ConA response was depressed at day 6 postchallenge. This systemic immunosuppression in susceptible avian hosts is in accordance with previous findings (40) and is a common feature of several parasitic infections (23). An unexpected finding was that in these same naive, challenged birds, the splenic ConA response appeared normal (Table 4). This result differs considerably from a previous report in which splenic lymphocyte responsiveness to another nonspecific Tcell mitogen was maximally depressed in naive birds at day 6 postinfection (40). Possible explanations for this discrepancy include differences in bird age, mitogen, and E. tenella strain used in the present study. The apparent difference between the local (spleen) and systemic (PBL) mitogenic responses in naive, infected birds may be a more general indication of specific alterations in lymphocyte migratory events which transpire following antigenic insult (41, 42). Acquired immunity to Eimeria is associated with the developing intracellular asexual stages (for a review, see reference 45). In an immune host, sporozoites will invade target gut cells but are prevented from continued development (24). Moreover, birds orally vaccinated with X-irradiated oocysts are protected against challenge, and sporozoites in the gut epithelial cells in these animals fail to exhibit significant merogonic development (16). The isolation and biochemical characterization of these intracellular parasite metabolic products have been hampered by the inability to reproducibly grow Eimeria spp. to high densities in vitro. Our laboratory has partially

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addressed this problem through the identification of an established cell line, SB-CEV-1/F7, which supports vigorous E. tenella sporozoite invasion and merozoite production (9). Results presented herein indicate that parasite antigens contained in these infected SB-CEV-1/F7 tissue culture supernatants stimulate PBL and splenic lymphocytes from birds previously exposed to E. tenella. Since these infected tissue culture supernatants can be quantified by ELISA using rabbit polyclonal antisporozoite sera, parasite proteins present in these infected supernatants share cross-reactive epitopes with sporozoites. Furthermore, 1-day-old B19B19 birds vaccinated with infected SB-CEV-1/F7 72-h tissue culture supernatants are partially protected against clinical disease (3). Preliminary data also suggest that E. tenella parasite proteins present in these supernatants obtained from infected tissue culture can be successfully fractionated and screened for the ability to stimulate immune lymphocytes isolated from naturally exposed birds (2). In an attempt to determine an optimal postchallenge time point at which to discriminate a protective immune response in resistant birds from a nonprotective immune response in susceptible birds, PBL responses to extracellular and tissue culture-derived parasite antigens were assayed at various days postchallenge in naive and trickle-immunized birds. Results suggest that there may be a differential PBL response between protected and susceptible birds at day 4 postchallenge to sporozoite and merozoite antigens. Birds previously exposed to oocysts by natural infection, when rechallenged, developed low but detectable parasite-specific responses earlier than naive birds following oocyst challenge. In summary, our laboratory has developed an immune model for avian coccidiosis in serologically defined B-locus young chicks. More specifically, trickle-immunized 1-day-old B19B19 birds can be used as a model to study host protective immune responses to in vitro-produced coccidial antigens. The use of PBL obtained from trickle-immunized B19B19 birds challenged at 10 days of age will facilitate the in vitro screening of biochemically fractionated E. tenella-infected SB-CEV-1/F7 72-h tissue culture supernatants. The development of bioassays for avian cytokines, such as gamma interferon, interleukin-2, and tumor necrosis factor, will aid in further understanding the host immune response to SB-CEV-1/F7 coccidial antigens. Unification of the immune model in a defined B-haplotype bird line with the production of E. tenella antigens in vitro should provide a rationale for the future identification, cloning, and expression of specific E. tenella genes for protective immunogens. ACKNOWLEDGMENTS We thank T. Banas and J. Lineberger for excellent technical assistance. We also thank P. Augustine and H. Danforth for critical review of the manuscript. REFERENCES 1. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254. 2. Brake, D. A. Unpublished data. 3. Brake, D. A., G. Strang, J. E. Lineberger, C. H. Fedor, R. Clare, T. A. Banas, and T. Miller. Immunogenic characterization of a tissue culture-derived vaccine which affords partial protection against avian coccidiosis. Submitted for publication. 4. Briles, W. E., W. H. McGibbon, and M. R. Irwin. 1950. On multiple alleles affecting cellular antigens in the chicken. Genetics 35:633–652. 5. Brothers, V. M., I. Kuhn, L. S. Paul, J. D. Gabe, W. H. Andrews, S. M. Sias, M. T. McCanan, E. A. Dragon, and J. G. Files. 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. 6. Bumstead, J. M., N. Bumstead, L. Rothwell, and F. M. Tomley. 1995. Comparison of immune responses in inbred lines of chickens to Eimeria maxima

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Editor: S. H. E. Kaufmann

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