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Responses of chickens to a recombinant refractile body antigen of Eimeria tenella administered using various immunizing strategies
IMMUNOLOGY AND MOLECULAR BIOLOGY Responses of Chickens to a Recombinant Refractile Body Antigen of Eimeria tenella Administered Using Various Immunizing Strategies S. H. Kopko,* D. S. Martin,† J. R. Barta*,1 *Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1, and †Department of Viral, Parasitic, and Preventable Diseases, Ontario Ministry of Health, 81 Resources Road, Etobicoke, Ontario, Canada N9P 3T1 were determined and compared with those of the controls. Significant protection against cecal lesions and weight loss was noted in birds inoculated with oocysts or injected with pcDNA3-SO7′ (25 µg). Birds injected with other doses of pcDNA3-SO7′ (12.5, 50, 60, and 100 µg) showed no reduction in cecal lesions and maintained poor rates of growth similar to controls. The recombinant antigen (CheY-SO7′) was also nonprotective. These results offer promise to the use of DNA immunization strategies for controlling avian coccidiosis and show for the first time naked DNA vaccination with a single coccidial antigen that protects chickens (as measured by reduced lesion scores and high rate of growth) against cecal coccidiosis.
(Key words: Eimeria tenella, immunization, naked DNA, recombinant antigen, refractile body antigen) 2000 Poultry Science 79:336–342
method was needed that preferentially elicited a cytotoxic T-lymphocyte response. Recent observations on the efficacy of DNA-based vaccine strategies (Bernd, 1997) and preliminary observations on their mode of stimulation of the immune response (Corr et al., 1996) have suggested that DNA-based vaccination might be such a method. One candidate antigen for the development of an anticoccidial DNA-based vaccine is SO7′ (Profous-Juchelka et al., 1988), found within refractile bodies of sporozoites and recognized by monoclonal antibody (mAb) 1209 (Danforth and Augustine, 1983). Indirect immunofluorescent antibody tests reveal that SO7′ contains an epitope common to all Eimeria species infecting the domestic fowl (Danforth and Augustine, 1983; Abrahamsen et al., 1994; Vermeulen, 1998) as well as to other coccidia including Lankesterella minima, a parasite of anurans (Herzenberg et al., 1995). The SO7′ gene has been sequenced and found to possess both B and T cell epitopes (Profous-Juchelka et al., 1988). It is highly immunogenic and elicits a strong antibody response in birds during natural infections (Tennyson, 1994). In this study, various forms of the SO7′ antigen (viable parasites, proteinaceous recombinant antigen, and plas-
INTRODUCTION Eimeria tenella is one of the four principal coccidial pathogens infecting domestic fowl in North America (Long and Reid, 1982). It is an obligate, intracellular protozoan parasite of chickens and is the cause of cecal coccidiosis. Since the 1950s, the poultry industry has used anticoccidial compounds to control this disease. However, the continued development of drug resistance (Chapman, 1986; Greif et al., 1996; Martin et al., 1997) has resulted in reexamination of other types of coccidial control. Birds rapidly become immune through natural infection; therefore, attempts have been made to develop live vaccines, attenuated vaccines, and, most recently, nonviable vaccines (Vermeulen, 1998). Nonviable vaccines have thus far failed to elicit protection comparable with that of live vaccination, perhaps because of improper presentation of antigen to the immune system (Danforth and Augustine, 1985; Crane et al., 1991; Danforth et al., 1993). Observations that cellular immunity may be central to successful protection against coccidial challenge (Rose and Hesketh, 1979; Lillehoj, 1987; Schito et al., 1996) suggested that a
Received for publication March 8, 1999. Accepted for publication October 15, 1999. 1 To whom correspondence should be addressed: [email protected].
Abbreviation Key: mAb = monoclonal antibody; Mr = Relative rate of migration.
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ABSTRACT A refractile body antigen (designated SO7′) found in sporozoites of Eimeria tenella was administered to chickens in various immunizing forms to assess its ability to protect against virulent challenge. These included native antigen from the parasite (viable oocysts; per os), recombinant antigen (CheY-SO7′; s.c.), and naked DNA immunization (pcDNA3-SO7′; i.m.). Male White Leghorn chicks were inoculated with oocysts or injected with one of the forms of SO7′ antigen at 1 wk of age, boosted at 3 wk of age with the same treatment, and subsequently challenged at 5 wk of age with oocysts of Eimeria tenella USDA Strain 80. Seven days postchallenge, chicks were killed, and weight gains and lesion scores
IMMUNIZING STRATEGIES USING A REFRACTILE BODY ANTIGEN
mid-encoded antigen) were administered to chickens. These immunization strategies were assessed biologically (via cecal lesion scores and growth performance) for their ability to protect against oral challenge with a virulent strain of E. tenella.
MATERIALS AND METHODS Monoclonal Antibody 1209 The cloned murine monoclonal cell line that secretes monoclonal antibody 1209 (subclass IgG2a) was provided by H. D. Danforth, USDA, Beltsville, MD 20705 (see Danforth and Augustine, 1983).
Propagation of Eimeria tenella Oocysts. BarredRock × White Leghorn chicken crosses (University of Guelph strain) 6 to 9 wk of age were used to obtain oocysts of Eimeria tenella USDA Strain 80. Birds were raised free of coccidia and provided access ad libitum to food and water. Determination of infective doses of parasite, recovery of oocysts from infected chickens, and subsequent sporulation of oocysts were conducted according to the protocol described by Long et al. (1976). CheY-SO7 ′—Fusion Protein Expression and Purification. By using a modification of the method described by Crane et al. (1991), the fusion protein CheY-SO7′ was expressed in Escherichia coli from the prokaryotic expression vector CheY-SO7′ [refer to Crane et al. (1991) for CheY-SO7′ map]. The LB broth containing ampicillin2 (Catalog No. 835-242) was inoculated with a single colony of bacteria, and the culture was grown until midlog growth was reached (A550 ≅ 0.5), at which time isopropylthio-β-D-galactoside (IPTG)2 (Catalog No. 724-815) was added to a final concentration of 100 µM. The broth was cultured for an additional 3 to 4 h and chilled on ice, and the bacteria were pelleted by centrifugation. Bacterial cells were disrupted by suspending the pellet in 10 mL of Buffer A [30 mM Tris2 (Catalog No. 708-968), pH 8.0, 5 mM EDTA2 (Catalog No. 808-261), 1.0 mM phenylmethylsulphonylfluoride (PMSF)3] and sonicating on ice at 2.5-min intervals for 10 min. The cell lysate was centrifuged (20,000 g, 45 min, 4 C; Sorvall centrifuge4 with a SS-34 rotor), and the pellet was resuspended in 10 mL Buffer A, to which 0.1 mL 10% Triton X-1003 (Catalog No. T-8532) was added. The suspension was stirred for
2
Boehringer Mannheim Canada, Laval, Quebec, Canada H7V 4A2. Sigma Chemical Co., St. Louis, MO 63178-9916. Model RC-5B, Mandel Scientific Company Ltd., Guelph, Ontario, Canada N1H 6J3. 5 Fisher Scientific, Pittsburgh, PA 15238. 6 Spectrum Medical Industries, Inc., Houston, TX 77073-4716. 7 Millipore (Canada) Ltd., Mississauga, Ontario, Canada L4V 1M5. 8 Bio-Rad Laboratories, Richmond, CA 94804. 9 PE Applied Biosystems, Mississauga, Ontario, Canada L5N 2M2. 10 Canadian Life Technologies, Burlington, Ontario, Canada L7P 1A1. 11 Eastman Kodak Company, Rochester, NY 14650. 3 4
60 min at 0 C in an ice bath and centrifuged as described above. The recombinant protein, contained in the pellet, was solubilized in 1.0 mL 6 M guanidine-HCl3 (Catalog No. G-7153) containing 15.4 mg dithiothreitol (DTT)3 (Catalog No. D-9163) and was placed on a heat block at 50 C with occasional shaking for 2 h. The solution was diluted with the addition of 10 mL of 7 M urea5 and centrifuged as described above. To 1-mL aliquots of the retained supernatant was added 3 mg of DTT dissolved in 3.0 mL 5 M guanidine-HCl in 0.5 M Tris-HCl buffer, adjusted to pH 8.6. The reaction solution was stirred for 2 h at ambient temperature and then mixed for 30 min with 200 mg of iodoacetic acid3 (Catalog No. I-4386); pH was adjusted to 8.5 with 3 M Tris base3 (Catalog No. T1503). To remove urea and guanidine-HCl the reaction system was dialyzed (cellulose ester membrane; MWCO: 1,000, Spectra/Por威,6) against 50 mM ammonium bicarbonate3 (Catalog No. A-6141) with several buffer changes over 48 h at 4 C. The retentate was concentrated using Centrifugal Ultrafree-20 Filter units,7 and purified recombinant antigen was stored at −20 C until use. The above protocol was also used to purify the protein CheY from a control CheY plasmid lacking the coccidial cDNA insert. Western Blot and Silver Stain Analysis. The purified CheY-SO7′ fusion protein was lysed by boiling for 5 min in reducing loading buffer [LB; 0.2 mgⴢmL−1 Pyronin-Y5 (Catalog No. 773-384); 2% β-mercaptoethanol5 (Catalog No. 03446-100); 10% SDS8 (Catalog No. 161-0301); 0.5 M Tris-HCl, pH 6.8; 0.5 M EDTA; and 10% glycerol3 (Catalog No. G-5516)] prior to separation on a 10% gradient SDSPAGE gel, based on the method of Laemmli (1970). The separated antigens were electrophoretically transferred onto a polyvinylidene fluoride membrane (ImmobilonP7 Catalog No. IPVH 00010) as described by Towbin et al. (1979). Immunodetection was conducted using the Western-Light威 CSPD-based chemiluminescent protocol for polyvinylidene fluoride membranes (provided by the CSPD manufacturer).The blotted membrane was washed three times for 15 min in wash buffer [0.1% Tween-208 (Catalog No. 170-6531) in PBS, pH 7.4] and then placed in blocking buffer (wash buffer with 0.2% I-Block reagent9 Catalog No. AI300) for 30 min. For 30 min, the membrane was exposed to a 1:1,000 dilution of monoclonal antibody (mAb) 1209 in 50:50 wash:blocking buffer. After washing in wash buffer, an alkaline phosphatase-labeled goat antimouse IgG10 (1:10,000 dilution) was added to the membrane for 15 min. The membrane was washed and incubated in chemiluminescent substrate for alkaline phosphatase (CSPD-Disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2′-(5′-chloro)- tricyclo[3.3.1.13,7]decan]-4-yl)phenyl phosphate9 (Catalog No. CD010) substrate solution for 5 min and exposed to radiographic XAR5 Imaging film (Kodak威 Catalog No. 875 8252)11 until bands appeared. A replica of the gel prepared above was silver-stained according to the method of Peng et al. (1995). The purified CheY protein was also analyzed as above. Construction of the Recombinant DNA pcDNA3SO7 ′. The DNA from plasmid CheY-SO7′ was isolated
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Parasites
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Chicken Immunization Trials Male White Leghorn chickens (Shaver strain)18 were used in this study. Birds were obtained as day-old chicks and reared in the Class 2 OMAFRA (Ontario Ministry of Agriculture, Food and Rural Affairs) isolation facility of the University of Guelph. Feed and water were available ad libitum throughout the experiment. In a preliminary experiment (Experiment 1), 1-wk-old birds were randomly distributed by weight into eight groups of three
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I.D. Biotechnology Labs, London, Ontario, Canada N6A 5K2. Model 4050 Ultrospec威 II, LKB Biochrom Ltd., Cambridge, UK CB4 4FJ. 14 Amersham Pharmacia Biotech Inc., Baie D’urfe, Quebec, Canada H9X 3V1. 15 BIO 101 Inc., Vista, CA 92083. 16 Invitrogen威, Carlsbad, CA 92008. 17 Laboratory Services Division, University of Guelph, Ontario, Canada N1G 2W1. 18 Shaver Poultry Breeding Farms Ltd., Cambridge, Ontario, Canada N1R 5V9. 19 Gibco BRL Life Technologies, Inc., Gaithersburg, MD 20877. 13
birds to give an even weight distribution among groups (Gardiner and Wehr, 1950). Birds were placed in Horsfal units sterilized with ammonia, and each Horsfal unit was provided with filtered air. The 1-wk-old chickens were treated as follows: Group 1 birds acted as a negative control and were inoculated orally with PBS; Groups 2, 3, and 4 birds were injected i.m. (breast) with 25 µg, 60 µg, or 100 µg of pcDNA3-SO7′ in PBS, respectively. Group 5 birds acted as the negative control for pcDNA3-SO7′ and were injected i.m. with 100 µg of pcDNA3 in PBS; Group 6 birds were injected s.c. (nape of neck) with 1 µg of the purified antigen CheY (negative control for CheYSO7′) mixed with Freunds Incomplete Adjuvant19 (Catalog No. 15720-014) using a B-D Multifit Homogenizer. Group 7 birds were injected s.c. (nape of neck) with 1 µg of recombinant antigen CheY-SO7′ prepared in the same manner as that described for antigen given to Group 6 birds. The positive controls, Group 8 birds, were inoculated orally with 2,500 oocysts of E. tenella USDA Strain 80. All birds were checked for fecal oocyst production throughout the trial. Only the positive control birds (Group 8) shed any oocysts, confirming that none of the birds had been exposed to viable oocysts except birds of that group. At 2 wk postinoculation (p.i.), identical treatments were repeated for all birds in each group. Two wk after this second treatment, all birds were each challenged orally with 2,500 oocysts of E. tenella USDA Strain 80. Seven days after challenge, birds were killed, and their ceca were examined for coccidial lesions and scored from 0 to 4 according to the criteria described by Johnson and Reid (1970). On every seventh day throughout the experiment, individual body weights were obtained, starting at the day of first treatment, up to and including the termination of the experiment. For Experiment 2, inoculations, injections, and challenges were performed as described above. The doses of pcDNA3-SO7′ used were changed from 25, 60, and 100 µg to 12.5, 25, and 50 µg. Groups consisted of 10 birds each and were raised on the floor in disinfected rooms with feed and water provided ad libitum. Weight gains and cecal lesions were determined as before.
Statistical Analysis The weight gains and lesion scores from each experiment were subjected to analysis of variance using the protected LSD method (see Steel et al., 1997) to determine the effects of treatments. A probability of P < 0.05 was considered significant.
RESULTS Confirmation of Purified Fusion Protein CheY-SO7 ′ Using Western Blotting and Silver Staining Silver stain analysis of purified CheY-SO7′ revealed a fusion protein with relative rate of migration (Mr) indicating an appropriate molecular mass of 36 kDa (Crane et al.,
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and purified using the IDPure Plasmid Kit12 (Catalog No. ID1010100) as per the manufacturer’s instructions. The DNA was quantified using a UV/VISIBLE spectrophotometer.13 The purified DNA, CheY-SO7′, was cleaved using the restriction enzyme EcoRI14 (Catalog No. 270854), electrophoresed on a 1% agarose gel in 1× TAE buffer (40 mM Tris-HCl; 10 mM Na-Acetate3—Catalog No. S-8625; and 1 mM EDTA, pH 8.0) and stained with ethidium bromide3 (Catalog No. E-8751). The DNA band containing the SO7′ insert (approximately 980 bp) was excised from the gel and purified using the Geneclean II威 Kit15 (Catalog No. 1001-400) as per the manufacturer’s instructions. The mammalian expression vector, pcDNA3 (refer to Invitrogen web site: www.invitrogen.com), was used as the eukaryotic expression vector. The plasmid (5.4 kb) was isolated, cleaved with EcoRI, and purified using the protocols described above for CheY-SO7′. The SO7′ and pcDNA3 fragments were ligated using the Rapid DNA Ligation Kit2 (Catalog No. 1635379) as per manufacturer’s instructions. Competent bacterial cells Strain 10F′16 (Catalog No. C665-03) were transformed with this recombinant plasmid by following a standard protocol (Sambrook et al., 1989). To ensure that the SO7′ insert was in the proper orientation and reading frame, clones containing inserts of the appropriate size were sequenced on an ABI Prism 377 Automated DNA Sequencer by Guelph Molecular Supercentre.17 The T7 universal primer was used as the forward primer, and 5′-AGAAGGCACAGTCGAGGCTG-3′ was used as the reverse sequence primer to obtain the sequence of the insert in both directions. Large-scale production of purified pcDNA3-SO7′ was performed using the PurePrep Macro Plasmid Purification Kit14 (Catalog No. 27-5210-01) as per manufacturer’s instructions, and purified plasmid was stored at −20 C.
IMMUNIZING STRATEGIES USING A REFRACTILE BODY ANTIGEN
1991). Western blot transfer and immunological detection using mAb 1209 (primary antibody) and alkaline phosphatase-conjugated antibody (secondary antibody) enabled detection of purified fusion protein CheY-SO7′. A band with an Mr of approximately 36 kDa reacted strongly with mAb 1209 (data not shown). Silver-stained gels containing electrophoretically separated samples of purified CheY demonstrated a few weakly stained bands. One of these had an Mr of appropriately 13 kDa, consistent with the CheY recombinant protein (Crane et al., 1991). Monoclonal antibody 1209 did not react with the purified CheY protein on Western blot analysis (data not shown).
Protective Immunity Against Coccidiosis
FIGURE 1. Mean lesion scores in the ceca of chickens at 7 d postchallenge with 2.5 × 103 oocysts of Eimeria tenella USDA Strain 80. All chickens were treated 14 and 28 d prior to challenge with their respective treatment types as indicated. Figure 1A illustrates mean lesion scores from three chickens of each indicated treatment type. Figure 1B data represents mean lesion scores. The asterisks indicate the mean lesion scores that are significantly different (P < 0.05) from their respective controls.
FIGURE 2. Distribution of cecal lesion scores among chickens 7 d postchallenge with 2.5 × 103 oocysts of Eimeria tenella USDA Strain 80 during the second experiment. Open bars represent low lesion scores in the range of 0 to 1.9, hatched bars correspond to moderate lesion scores in the range of 2.0 to 2.9, and solid bars represent severe lesion scores in the range of 3.0 to 4.0. The y-axis indicates the number of birds in each group possessing cecal lesions of the indicated severity. All chickens had been treated 14 and 28 d prior to challenge with their respective treatment types as indicated on the graph.
least severe (averaging <1.0) in birds inoculated per os with oocysts or injected with low dose pcDNA3-SO7′ (25 µg) relative to their respective controls. In birds injected with higher doses of pcDNA3-SO7′, the lesion scores after challenge were more severe. Birds injected with the recombinant antigen (CheY-SO7′) had mean lesion scores ranging from 1.5 to 2.0, similar to those seen in the CheY control. Cecal lesion scores in the second study (Figure 1B) were similar to those seen in the preliminary trial. Lesion scores were least severe with the oocyst positive control group, which had a mean lesion score of 0 (significantly lower than the PBS control birds). Birds injected with the 25 µg pcDNA3-SO7′ had a mean lesion score (1.8) significantly less than the pcDNA3 control. All other groups had mean lesion scores >2 and were not significantly different from their respective controls. Figure 2 shows in detail the distribution of lesion severity among birds. The oocyst positive control group was well protected (100% of birds were free of lesions). In contrast, the majority (80%) of birds in the PBS negative control group demonstrated moderate (lesion score of 2.0 to 2.9) to severe (lesion score of 3.0 to 4.0) lesions. In the 12.5 µg pcDNA3-SO7′ group, all of the birds developed lesion scores ≥2.0. However, the majority (50%) of birds injected with the 25 µg dose showed only minor damage to their cecal mucosal tissue, as evidenced by lesion scores <2.0. The 50 µg pcDNA3SO7′ group followed the trend observed for the first set of experiments in which protection decreased with larger doses of pcDNA3-SO7′ (80% of birds developed lesion scores ≥2.0). Some evidence of protection was found in the CheY-SO7′-treated chickens; 40% had lesions <2.0. The corresponding CheY-treated control birds had lesions ≥2.0 in 80% of the birds. For both experiments, variances did not differ significantly between groups. Chicken Weight Gains. Average daily weight gain (grams) in chickens from day of challenge to 7 d following challenge are summarized in Table 1 for each treatment.
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Lesion Scores. In the preliminary experiment, cecal lesions evaluated at 7 d postchallenge (Figure 1A) were
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KOPKO ET AL. TABLE 1. Average daily weight gain (grams) in chickens from day of challenge to 7 d postchallenge during Experiment 11
Group
Number
Treatment
Average daily gain (g) ±SE
1 2 3 4 5 6 7 8
3 3 3 3 3 3 3 3
PBS (1 mL per os) pcDNA3-SO7′ (25 µg i.m. breast) pcDNA3-SO7′(60 µg i.m. breast) pcDNA3-SO7′ (100 µg i.m. breast) pcDNA3 (100 µg i.m. breast) CheY (1 µg s.c.) CheY-SO7′ (1 µg s.c.) Oocyst (2.5 × 103 per os)
16.71 18.09 17.29 18.29 16.33 15.33 13.71 17.62
± ± ± ± ± ± ± ±
0.459 0.504 1.784 1.692 0.585 0.826 1.622 0.882
1 Chickens were treated at 1 wk and 3 wk of age, and were challenged at 5 wk of age with 2.5 × 103 oocysts of Eimeria tenella USDA Strain 80.
DISCUSSION Experiments were conducted to assess the effects of the route of administration and form of a single coccidial antigen (refractile body antigen SO7′) on the resulting immune response of birds. This response was measured using biological indicators of immunological protection against coccidial challenge. Specifically, birds were challenged with virulent parasites, and the rate of growth and degree of protection (if any) from cecal lesions were determined. Other than natural infections induced by oocysts administered per os, intramuscular injection with the 25-µg
dose of plasmid DNA containing the SO7′ insert under the control of a viral promoter induced the greatest level of protection against challenge with E. tenella infection. In both experiments, lesion scores were reduced to 0 to 1.5 in more than 50% of the chickens. In comparison, 90% of the control chickens showed a moderate to severe degree of infection (lesion score range 2.0 to 4.0). Decreased damage to the cecal mucosa in chickens injected with 25 µg pcDNA3-SO7′ suggests the involvement of an immune effector mechanism that may have resulted in attrition or inhibition of development of early stages of the parasite life cycle such as sporozoites or first-generation merogonic stage. The inability of this plasmid DNA to significantly decrease lesion scores in 100% of the vaccinated subjects may be attributed to variability in expression of the recombinant antigen among birds in a single experiment and between experiments. Chickens injected with 12.5 µg pcDNA3-SO7′ and high doses of pcDNA3-SO7′ (50, 60, or 100 µg) demonstrated severe lesions comparable with those of the pcDNA3 control (100 µg). Previous immunization studies (H. ProfousJulchelka and M. Hozza, 1993, Merck & Co., Inc., PO Box 2000, R8OY-265, Rahway, NJ 07065, personal communication) have found that multiple injections of 100 µg of plasmid DNA (RSV expression vector containing SO7′ gene) did not elicit protection against lesion scores in challenge infections. A possible explanation for the lack
TABLE 2. Average daily weight gain (grams) in chickens from day of challenge to 7 d postchallenge during Experiment 21
Group
Number
Treatment
Average daily gain (g) ±SE
1 2 3 4 5 6 7 8
8 8 10 9 7 7 7 10
PBS (1 mL per os) pcDNA3-SO7′ (12.5 µg i.m. breast) pcDNA3-SO7′ (25 µg i.m. breast) pcDNA3-SO7′ (50 µg i.m. breast) pcDNA3 (50 µg i.m. breast) CheY (1 µg s.c.) CheY-SO7′ (1 µg s.c.) Oocysts (2.5 × 103 per os)
11.57 10.95 13.48 12.90 9.97 13.53 13.98 18.97
± ± ± ± ± ± ± ±
0.979 1.307 0.8152 1.200 1.302 1.041 1.003 0.8683
1 Chickens were treated at 1 wk and 3 wk of age, and were challenged at 5 wk of age with 2.5 × 103 oocysts of Eimeria tenella USDA Strain 80. 2 Significantly different from the pcDNA3 control birds (Group 5) at P < 0.05. 3 Significantly different from the PBS control birds (Group 1) and all other treatment groups at P < 0.05.
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The average daily weight gain was greatest in birds inoculated with 25- and 100-µg doses of pcDNA3-SO7′; however, these birds did not demonstrate rates of growth that were significantly greater than that of the pcDNA3 control group. The small sample size resulted in no significant differences between any groups in the first experiment. In the second experiment (Table 2), birds treated with oocysts or the 25-µg dose of pcDNA3-SO7′ showed significantly higher average daily weight gains than did the controls (PBS and pcDNA3, respectively). The average daily weight gain of birds injected with recombinant antigen (CheY-SO7′) was not significantly higher than the CheY-negative control group.
IMMUNIZING STRATEGIES USING A REFRACTILE BODY ANTIGEN
means of immunizing chickens against challenge infections with E. tenella. However, our study demonstrated that a carefully titrated dose of a refractile body antigen delivered to chickens in a DNA-based vaccine construct was able to elicit an immune response that provided significant biological protection (as measured by lesion score reduction and maintained high rate of growth) against virulent E. tenella challenge. This result is the first demonstration of naked DNA vaccination that works in chickens against cecal coccidiosis. These results provide encouragement that cloned coccidial antigens, singly or more likely in combination, may be included in a commercially viable, subunit vaccine against avian coccidiosis (Jenkins, 1998; Vermeulen, 1998). The lack of efficacy of the previous attempts at vaccination using recombinant antigens may be more related to the form of the antigen and its route of delivery to the birds than to the intrinsic immunogenicity of these recombinant coccidial proteins. The DNA vaccination may be preferable to proteinaceous antigens because of the relative ease of production, administration, and storage compared with recombinant proteins.
ACKNOWLEDGMENTS We thank Harry Danforth for providing us with the cell line that secretes mAb 1209; Helen Profous-Juchelka, Julie Cobean, Susan Slack, Krystina Strickler, Muhammad Tahir, Cindy Kopko, and Sunny Mann for technical assistance; and Steve Gismondi and William Sears for their help with the statistical analyses. We also thank Aggie Fernando, Ramon Carreno, and Krystina Strickler for their criticisms of earlier drafts of this manuscript. This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada (106453-98) and the Ontario Ministry of Agriculture, Food and Rural Affairs Research Program to John R. Barta.
REFERENCES Abrahamsen, M. S, R. R. Johnson, T. G. Clark, and M. W. White, 1994. Developmental regulation of an Eimeria bovis mRNA encoding refractile body-associated protein. Mol. Biochem. Parasitol. 68:25–34. Augustine, P. C., and H. D. Danforth, 1986. A study of the dynamics of the invasion of immunized birds by Eimeria sporozoites. Avian Dis. 30:347–351. Bernd, H. K., 1997. DNA vaccines for parasitic infections. Immun. and Cell Biol. 75:370–375. Chapman, H. D, 1986. Drug resistance in coccidia: recent research. Pages 330–347 in: Research in Avian Coccidiosis. L. R. McDougald, L. P. Joyner, and P. L. Long, ed. Proc. Georgia Coccidiosis Conference, Athens, GA. Corr, M., D. J. Lee, D. A. Carson, and H. Tighe, 1996. Gene vaccination with naked plasmid DNA: Mechanism of CTL priming. J. Exp. Med. 184:1555–1560. Crane, M.S.J., B. Goggin, R. M. Pellegrino, O. J. Ravino, C. Lange, Y. D. Karkhanis, K. E. Kirk, and P. R. Chakraborty, 1991. Cross-protection against four species of chicken coccidia with a single recombinant antigen. Infect. Immun. 59:1271–1277. Danforth, H. D., and P. C. Augustine, 1983. Specificity and crossreactivity of immune serum and hybridoma antibodies to various species of avian coccidia. Poultry Sci. 62:2145–2151.
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of protection with the high doses of pcDNA3-SO7′ may be derived from studies on DNA vaccines in other systems. Mice immunized with different doses of a Plasmodium yoelli circumsporozoite protein DNA-based vaccine exhibited a dose-related response in which antibody levels increased with greater amounts of injected DNA (Gramzinski et al., 1997). By analogy, doses of pcDNA3SO7′ >25 µg may be inducing primarily an antibody response that has been shown to play only a minor role in protection against coccidial infection (Rose et al., 1984; Augustine and Danforth, 1986). The inability to protect chickens with the 12.5-µg dose of pcDNA3-SO7′ may be due to expression of the antigen at levels insufficient to elicit any immune response and would explain why lesions and weight gains were similar to those of the PBS controls. Most chickens treated with the recombinant antigen (CheY-SO7′) were not protected against cecal lesions. Past studies on immunization of chickens with CheY-SO7′ (Crane et al., 1991) showed that a single dose, without adjuvant, protected against severe coccidiosis induced by infection with E. tenella. In addition, chickens treated with CheY-SO7′ exhibited protection against challenge with heterologous species (Eimeria acervulina, Eimeria maxima, and Eimeria necatrix). However, the mean lesion score was never reduced below 1.5, indicating that immunization with the fusion protein CheY-SO7′ only elicited a low level of protection. In our studies, the dose of CheY-SO7′ (1 µg) was comparable with that used by Crane et al. (1991). According to their reports, increasing the dose of CheY-SO7′ would not have improved the observed protection in this study. Differences in the type of chicken used in the present experiment (White Leghorn layers vs production broilers) or antigen delivery (with or without adjuvant) may explain the differences in response to the same recombinant antigen observed in the present experiments and by Crane et al. (1991). Another biological measure used to assess protection against coccidial infection in chickens is weight gain following challenge. Protection against coccidial challenge is indicated by a high average daily weight gain as seen with the oocyst group, which was immunized multiple times with the parasite throughout the experiment. The negative control group (PBS) had a significantly lower rate of growth than the oocyst-positive control in the second immunization trials. The only experimental group to exhibit weight gain significantly greater than their negative control group was the 25 µg pcDNA3-SO7′ treatment group in the second set of experiments. Again, treatment with the 25 µg pcDNA3-SO7′ induced an intermediate response between pcDNA3 (negative control) and oocyst (positive control) treatments, as was seen with lesion scores. The only study with a coccidial subunit vaccine to demonstrate some degree of protection against both weight loss and lesion scores in birds was one using a fusion protein containing a merozoite surface antigen (MZ250) (Jenkins et al., 1991). Overall, these immunization trials demonstrate that the viable parasite form (oocyst) is still the most effective
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KOPKO ET AL. Long, P. L., and W. M. Reid, 1982. A guide for the diagnosis of coccidiosis in chickens. Research Report 404. University of Georgia, Athens, GA. Martin, A. G., H. D. Danforth, J. R. Barta, and M. A. Fernando. 1997. Analysis of immunological cross-protection and sensitivities to anticoccidial drugs among five geographical and temporal strains of Eimeria maxima. Int. J. Parasitol. 5:527–533. Peng, H., M. Du, J. Ji, P. G. Isaacson, and L. Pan, 1995. Highresolution SSCP analysis using polyacrylamide agarose composite gel and a background-free silver staining method. Biotechniques 19:410–414. Profous-Juchelka, H., P. Liberator, and M. J. Turner, 1988. Identification and characterization of cDNA clones encoding antigens of Eimeria tenella. Mol. Biochem. Parasitol. 30:233–242. Rose, M. E., and P. Hesketh, 1979. Immunity to coccidiosis: Tlymphocyte or B-lymphocyte deficient animals. Infect. Immun. 26:630–637. Rose, M. E., A. M. Lawn, and B. J. Millard, 1984. The effect of immunity on the early events in the life-cycle of Eimeria tenella in the caecal mucosa of the chicken. Parasitology 88:199–210. Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Schito, M. L., J. R. Barta, and B. Chobotar, 1996. Comparison of four murine Eimeria species in immunocompetent and immunodeficient mice. J. Parasitol. 82:255–262. Steel, R.G.D., J. H. Torrie, and D. A. Dicky, 1997. Page 178 in: Principles and Procedures of Statistics: A Biometrical Approach. 3rd ed. The McGraw Hill Companies Inc., New York, NY. Tennyson, S. A., 1994. Investigation of a low molecular weight antigen of Eimeria tenella. M.S. Thesis, University of Guelph, Guelph, Canada. Towbin, H., T. Staehelin, and J. Gordon, 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets-procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350–4354. Vermeulen, A. N., 1998. Progress in recombinant vaccine development against coccidiosis. A review and prospects into the next millennium. Int. J. Parasitol. 28:1121–1130.
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Danforth, H. D., and P. C. Augustine, 1985. Use of hybridoma antibodies and recombinant DNA technology in protozoan vaccine development. Avian Dis. 30:37–42. Danforth, H. D., P. C. Augustine, and M. C. Jenkins, 1993. A review of progress in coccidial vaccine development. Pages 49–60 in: VIth International Coccidiosis Conference, Guelph, Ontario, Canada. Gardiner, J. L., and E. E. Wehr, 1950. Selecting experimental groups of chicks by weight. Proc. Helminthological Soc. Washington 17:25–26. Gramzinski, R. A., C. M. Dorina, D. Doolan, Y. Charoenvit, N. Obaldia, R. Rossan, M. Sedegah, R. Wang, P. Hobart, M. Margalith, and S. Hoffman, 1997. Malaria DNA vaccines in Aotus monkeys. Vaccine 15:913–915. Greif, G., B. Stephan, and A. Haberkorn, 1996. Intraspecific polymorphisms of Eimeria species due to resistance against anticoccidial drugs. Parasitol. Res. 82:706–714. Herzenberg, A. M., J. R. Barta, and S. S. Desser, 1995. Monoclonal antibodies raised against coccidia and malarial parasites recognize antigenic epitopes found in lankesterellid and adeleorin parasites. J. Parasitol. 81:543–548. Jenkins, M. C., 1998. Progress on developing a recombinant coccidiosis vaccine. Int. J. Parasitol. 28:1111–1119. Jenkins, M. C., M. D. Castle, and H. D. Danforth, 1991. Protective immunization against the intestinal parasite Eimeria acervulina with recombinant coccidial antigen. Poultry Sci. 70:539–547. Johnson, J. K., and W. M. Reid, 1970. Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments with chickens. Exp. Parasitol. 28:30–36. Laemmli, U. K., 1970. Cleavage of structural proteins during assembly of the head of the bacteriophage T4. Nature 227:680–685. Lillehoj, H. S., 1987. Effects of immunosuppression on avian coccidiosis. Cyclosporin A but not hormonal bursectomy abrogates host protective immunity. Infect. Immun. 55:1616– 1621. 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. 6:201–207.
(Key words: Eimeria tenella, immunization, naked DNA, recombinant antigen, refractile body antigen) 2000 Poultry Science 79:336–342
method was needed that preferentially elicited a cytotoxic T-lymphocyte response. Recent observations on the efficacy of DNA-based vaccine strategies (Bernd, 1997) and preliminary observations on their mode of stimulation of the immune response (Corr et al., 1996) have suggested that DNA-based vaccination might be such a method. One candidate antigen for the development of an anticoccidial DNA-based vaccine is SO7′ (Profous-Juchelka et al., 1988), found within refractile bodies of sporozoites and recognized by monoclonal antibody (mAb) 1209 (Danforth and Augustine, 1983). Indirect immunofluorescent antibody tests reveal that SO7′ contains an epitope common to all Eimeria species infecting the domestic fowl (Danforth and Augustine, 1983; Abrahamsen et al., 1994; Vermeulen, 1998) as well as to other coccidia including Lankesterella minima, a parasite of anurans (Herzenberg et al., 1995). The SO7′ gene has been sequenced and found to possess both B and T cell epitopes (Profous-Juchelka et al., 1988). It is highly immunogenic and elicits a strong antibody response in birds during natural infections (Tennyson, 1994). In this study, various forms of the SO7′ antigen (viable parasites, proteinaceous recombinant antigen, and plas-
INTRODUCTION Eimeria tenella is one of the four principal coccidial pathogens infecting domestic fowl in North America (Long and Reid, 1982). It is an obligate, intracellular protozoan parasite of chickens and is the cause of cecal coccidiosis. Since the 1950s, the poultry industry has used anticoccidial compounds to control this disease. However, the continued development of drug resistance (Chapman, 1986; Greif et al., 1996; Martin et al., 1997) has resulted in reexamination of other types of coccidial control. Birds rapidly become immune through natural infection; therefore, attempts have been made to develop live vaccines, attenuated vaccines, and, most recently, nonviable vaccines (Vermeulen, 1998). Nonviable vaccines have thus far failed to elicit protection comparable with that of live vaccination, perhaps because of improper presentation of antigen to the immune system (Danforth and Augustine, 1985; Crane et al., 1991; Danforth et al., 1993). Observations that cellular immunity may be central to successful protection against coccidial challenge (Rose and Hesketh, 1979; Lillehoj, 1987; Schito et al., 1996) suggested that a
Received for publication March 8, 1999. Accepted for publication October 15, 1999. 1 To whom correspondence should be addressed: [email protected].
Abbreviation Key: mAb = monoclonal antibody; Mr = Relative rate of migration.
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ABSTRACT A refractile body antigen (designated SO7′) found in sporozoites of Eimeria tenella was administered to chickens in various immunizing forms to assess its ability to protect against virulent challenge. These included native antigen from the parasite (viable oocysts; per os), recombinant antigen (CheY-SO7′; s.c.), and naked DNA immunization (pcDNA3-SO7′; i.m.). Male White Leghorn chicks were inoculated with oocysts or injected with one of the forms of SO7′ antigen at 1 wk of age, boosted at 3 wk of age with the same treatment, and subsequently challenged at 5 wk of age with oocysts of Eimeria tenella USDA Strain 80. Seven days postchallenge, chicks were killed, and weight gains and lesion scores
IMMUNIZING STRATEGIES USING A REFRACTILE BODY ANTIGEN
mid-encoded antigen) were administered to chickens. These immunization strategies were assessed biologically (via cecal lesion scores and growth performance) for their ability to protect against oral challenge with a virulent strain of E. tenella.
MATERIALS AND METHODS Monoclonal Antibody 1209 The cloned murine monoclonal cell line that secretes monoclonal antibody 1209 (subclass IgG2a) was provided by H. D. Danforth, USDA, Beltsville, MD 20705 (see Danforth and Augustine, 1983).
Propagation of Eimeria tenella Oocysts. BarredRock × White Leghorn chicken crosses (University of Guelph strain) 6 to 9 wk of age were used to obtain oocysts of Eimeria tenella USDA Strain 80. Birds were raised free of coccidia and provided access ad libitum to food and water. Determination of infective doses of parasite, recovery of oocysts from infected chickens, and subsequent sporulation of oocysts were conducted according to the protocol described by Long et al. (1976). CheY-SO7 ′—Fusion Protein Expression and Purification. By using a modification of the method described by Crane et al. (1991), the fusion protein CheY-SO7′ was expressed in Escherichia coli from the prokaryotic expression vector CheY-SO7′ [refer to Crane et al. (1991) for CheY-SO7′ map]. The LB broth containing ampicillin2 (Catalog No. 835-242) was inoculated with a single colony of bacteria, and the culture was grown until midlog growth was reached (A550 ≅ 0.5), at which time isopropylthio-β-D-galactoside (IPTG)2 (Catalog No. 724-815) was added to a final concentration of 100 µM. The broth was cultured for an additional 3 to 4 h and chilled on ice, and the bacteria were pelleted by centrifugation. Bacterial cells were disrupted by suspending the pellet in 10 mL of Buffer A [30 mM Tris2 (Catalog No. 708-968), pH 8.0, 5 mM EDTA2 (Catalog No. 808-261), 1.0 mM phenylmethylsulphonylfluoride (PMSF)3] and sonicating on ice at 2.5-min intervals for 10 min. The cell lysate was centrifuged (20,000 g, 45 min, 4 C; Sorvall centrifuge4 with a SS-34 rotor), and the pellet was resuspended in 10 mL Buffer A, to which 0.1 mL 10% Triton X-1003 (Catalog No. T-8532) was added. The suspension was stirred for
2
Boehringer Mannheim Canada, Laval, Quebec, Canada H7V 4A2. Sigma Chemical Co., St. Louis, MO 63178-9916. Model RC-5B, Mandel Scientific Company Ltd., Guelph, Ontario, Canada N1H 6J3. 5 Fisher Scientific, Pittsburgh, PA 15238. 6 Spectrum Medical Industries, Inc., Houston, TX 77073-4716. 7 Millipore (Canada) Ltd., Mississauga, Ontario, Canada L4V 1M5. 8 Bio-Rad Laboratories, Richmond, CA 94804. 9 PE Applied Biosystems, Mississauga, Ontario, Canada L5N 2M2. 10 Canadian Life Technologies, Burlington, Ontario, Canada L7P 1A1. 11 Eastman Kodak Company, Rochester, NY 14650. 3 4
60 min at 0 C in an ice bath and centrifuged as described above. The recombinant protein, contained in the pellet, was solubilized in 1.0 mL 6 M guanidine-HCl3 (Catalog No. G-7153) containing 15.4 mg dithiothreitol (DTT)3 (Catalog No. D-9163) and was placed on a heat block at 50 C with occasional shaking for 2 h. The solution was diluted with the addition of 10 mL of 7 M urea5 and centrifuged as described above. To 1-mL aliquots of the retained supernatant was added 3 mg of DTT dissolved in 3.0 mL 5 M guanidine-HCl in 0.5 M Tris-HCl buffer, adjusted to pH 8.6. The reaction solution was stirred for 2 h at ambient temperature and then mixed for 30 min with 200 mg of iodoacetic acid3 (Catalog No. I-4386); pH was adjusted to 8.5 with 3 M Tris base3 (Catalog No. T1503). To remove urea and guanidine-HCl the reaction system was dialyzed (cellulose ester membrane; MWCO: 1,000, Spectra/Por威,6) against 50 mM ammonium bicarbonate3 (Catalog No. A-6141) with several buffer changes over 48 h at 4 C. The retentate was concentrated using Centrifugal Ultrafree-20 Filter units,7 and purified recombinant antigen was stored at −20 C until use. The above protocol was also used to purify the protein CheY from a control CheY plasmid lacking the coccidial cDNA insert. Western Blot and Silver Stain Analysis. The purified CheY-SO7′ fusion protein was lysed by boiling for 5 min in reducing loading buffer [LB; 0.2 mgⴢmL−1 Pyronin-Y5 (Catalog No. 773-384); 2% β-mercaptoethanol5 (Catalog No. 03446-100); 10% SDS8 (Catalog No. 161-0301); 0.5 M Tris-HCl, pH 6.8; 0.5 M EDTA; and 10% glycerol3 (Catalog No. G-5516)] prior to separation on a 10% gradient SDSPAGE gel, based on the method of Laemmli (1970). The separated antigens were electrophoretically transferred onto a polyvinylidene fluoride membrane (ImmobilonP7 Catalog No. IPVH 00010) as described by Towbin et al. (1979). Immunodetection was conducted using the Western-Light威 CSPD-based chemiluminescent protocol for polyvinylidene fluoride membranes (provided by the CSPD manufacturer).The blotted membrane was washed three times for 15 min in wash buffer [0.1% Tween-208 (Catalog No. 170-6531) in PBS, pH 7.4] and then placed in blocking buffer (wash buffer with 0.2% I-Block reagent9 Catalog No. AI300) for 30 min. For 30 min, the membrane was exposed to a 1:1,000 dilution of monoclonal antibody (mAb) 1209 in 50:50 wash:blocking buffer. After washing in wash buffer, an alkaline phosphatase-labeled goat antimouse IgG10 (1:10,000 dilution) was added to the membrane for 15 min. The membrane was washed and incubated in chemiluminescent substrate for alkaline phosphatase (CSPD-Disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2′-(5′-chloro)- tricyclo[3.3.1.13,7]decan]-4-yl)phenyl phosphate9 (Catalog No. CD010) substrate solution for 5 min and exposed to radiographic XAR5 Imaging film (Kodak威 Catalog No. 875 8252)11 until bands appeared. A replica of the gel prepared above was silver-stained according to the method of Peng et al. (1995). The purified CheY protein was also analyzed as above. Construction of the Recombinant DNA pcDNA3SO7 ′. The DNA from plasmid CheY-SO7′ was isolated
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Parasites
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KOPKO ET AL.
Chicken Immunization Trials Male White Leghorn chickens (Shaver strain)18 were used in this study. Birds were obtained as day-old chicks and reared in the Class 2 OMAFRA (Ontario Ministry of Agriculture, Food and Rural Affairs) isolation facility of the University of Guelph. Feed and water were available ad libitum throughout the experiment. In a preliminary experiment (Experiment 1), 1-wk-old birds were randomly distributed by weight into eight groups of three
12
I.D. Biotechnology Labs, London, Ontario, Canada N6A 5K2. Model 4050 Ultrospec威 II, LKB Biochrom Ltd., Cambridge, UK CB4 4FJ. 14 Amersham Pharmacia Biotech Inc., Baie D’urfe, Quebec, Canada H9X 3V1. 15 BIO 101 Inc., Vista, CA 92083. 16 Invitrogen威, Carlsbad, CA 92008. 17 Laboratory Services Division, University of Guelph, Ontario, Canada N1G 2W1. 18 Shaver Poultry Breeding Farms Ltd., Cambridge, Ontario, Canada N1R 5V9. 19 Gibco BRL Life Technologies, Inc., Gaithersburg, MD 20877. 13
birds to give an even weight distribution among groups (Gardiner and Wehr, 1950). Birds were placed in Horsfal units sterilized with ammonia, and each Horsfal unit was provided with filtered air. The 1-wk-old chickens were treated as follows: Group 1 birds acted as a negative control and were inoculated orally with PBS; Groups 2, 3, and 4 birds were injected i.m. (breast) with 25 µg, 60 µg, or 100 µg of pcDNA3-SO7′ in PBS, respectively. Group 5 birds acted as the negative control for pcDNA3-SO7′ and were injected i.m. with 100 µg of pcDNA3 in PBS; Group 6 birds were injected s.c. (nape of neck) with 1 µg of the purified antigen CheY (negative control for CheYSO7′) mixed with Freunds Incomplete Adjuvant19 (Catalog No. 15720-014) using a B-D Multifit Homogenizer. Group 7 birds were injected s.c. (nape of neck) with 1 µg of recombinant antigen CheY-SO7′ prepared in the same manner as that described for antigen given to Group 6 birds. The positive controls, Group 8 birds, were inoculated orally with 2,500 oocysts of E. tenella USDA Strain 80. All birds were checked for fecal oocyst production throughout the trial. Only the positive control birds (Group 8) shed any oocysts, confirming that none of the birds had been exposed to viable oocysts except birds of that group. At 2 wk postinoculation (p.i.), identical treatments were repeated for all birds in each group. Two wk after this second treatment, all birds were each challenged orally with 2,500 oocysts of E. tenella USDA Strain 80. Seven days after challenge, birds were killed, and their ceca were examined for coccidial lesions and scored from 0 to 4 according to the criteria described by Johnson and Reid (1970). On every seventh day throughout the experiment, individual body weights were obtained, starting at the day of first treatment, up to and including the termination of the experiment. For Experiment 2, inoculations, injections, and challenges were performed as described above. The doses of pcDNA3-SO7′ used were changed from 25, 60, and 100 µg to 12.5, 25, and 50 µg. Groups consisted of 10 birds each and were raised on the floor in disinfected rooms with feed and water provided ad libitum. Weight gains and cecal lesions were determined as before.
Statistical Analysis The weight gains and lesion scores from each experiment were subjected to analysis of variance using the protected LSD method (see Steel et al., 1997) to determine the effects of treatments. A probability of P < 0.05 was considered significant.
RESULTS Confirmation of Purified Fusion Protein CheY-SO7 ′ Using Western Blotting and Silver Staining Silver stain analysis of purified CheY-SO7′ revealed a fusion protein with relative rate of migration (Mr) indicating an appropriate molecular mass of 36 kDa (Crane et al.,
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and purified using the IDPure Plasmid Kit12 (Catalog No. ID1010100) as per the manufacturer’s instructions. The DNA was quantified using a UV/VISIBLE spectrophotometer.13 The purified DNA, CheY-SO7′, was cleaved using the restriction enzyme EcoRI14 (Catalog No. 270854), electrophoresed on a 1% agarose gel in 1× TAE buffer (40 mM Tris-HCl; 10 mM Na-Acetate3—Catalog No. S-8625; and 1 mM EDTA, pH 8.0) and stained with ethidium bromide3 (Catalog No. E-8751). The DNA band containing the SO7′ insert (approximately 980 bp) was excised from the gel and purified using the Geneclean II威 Kit15 (Catalog No. 1001-400) as per the manufacturer’s instructions. The mammalian expression vector, pcDNA3 (refer to Invitrogen web site: www.invitrogen.com), was used as the eukaryotic expression vector. The plasmid (5.4 kb) was isolated, cleaved with EcoRI, and purified using the protocols described above for CheY-SO7′. The SO7′ and pcDNA3 fragments were ligated using the Rapid DNA Ligation Kit2 (Catalog No. 1635379) as per manufacturer’s instructions. Competent bacterial cells Strain 10F′16 (Catalog No. C665-03) were transformed with this recombinant plasmid by following a standard protocol (Sambrook et al., 1989). To ensure that the SO7′ insert was in the proper orientation and reading frame, clones containing inserts of the appropriate size were sequenced on an ABI Prism 377 Automated DNA Sequencer by Guelph Molecular Supercentre.17 The T7 universal primer was used as the forward primer, and 5′-AGAAGGCACAGTCGAGGCTG-3′ was used as the reverse sequence primer to obtain the sequence of the insert in both directions. Large-scale production of purified pcDNA3-SO7′ was performed using the PurePrep Macro Plasmid Purification Kit14 (Catalog No. 27-5210-01) as per manufacturer’s instructions, and purified plasmid was stored at −20 C.
IMMUNIZING STRATEGIES USING A REFRACTILE BODY ANTIGEN
1991). Western blot transfer and immunological detection using mAb 1209 (primary antibody) and alkaline phosphatase-conjugated antibody (secondary antibody) enabled detection of purified fusion protein CheY-SO7′. A band with an Mr of approximately 36 kDa reacted strongly with mAb 1209 (data not shown). Silver-stained gels containing electrophoretically separated samples of purified CheY demonstrated a few weakly stained bands. One of these had an Mr of appropriately 13 kDa, consistent with the CheY recombinant protein (Crane et al., 1991). Monoclonal antibody 1209 did not react with the purified CheY protein on Western blot analysis (data not shown).
Protective Immunity Against Coccidiosis
FIGURE 1. Mean lesion scores in the ceca of chickens at 7 d postchallenge with 2.5 × 103 oocysts of Eimeria tenella USDA Strain 80. All chickens were treated 14 and 28 d prior to challenge with their respective treatment types as indicated. Figure 1A illustrates mean lesion scores from three chickens of each indicated treatment type. Figure 1B data represents mean lesion scores. The asterisks indicate the mean lesion scores that are significantly different (P < 0.05) from their respective controls.
FIGURE 2. Distribution of cecal lesion scores among chickens 7 d postchallenge with 2.5 × 103 oocysts of Eimeria tenella USDA Strain 80 during the second experiment. Open bars represent low lesion scores in the range of 0 to 1.9, hatched bars correspond to moderate lesion scores in the range of 2.0 to 2.9, and solid bars represent severe lesion scores in the range of 3.0 to 4.0. The y-axis indicates the number of birds in each group possessing cecal lesions of the indicated severity. All chickens had been treated 14 and 28 d prior to challenge with their respective treatment types as indicated on the graph.
least severe (averaging <1.0) in birds inoculated per os with oocysts or injected with low dose pcDNA3-SO7′ (25 µg) relative to their respective controls. In birds injected with higher doses of pcDNA3-SO7′, the lesion scores after challenge were more severe. Birds injected with the recombinant antigen (CheY-SO7′) had mean lesion scores ranging from 1.5 to 2.0, similar to those seen in the CheY control. Cecal lesion scores in the second study (Figure 1B) were similar to those seen in the preliminary trial. Lesion scores were least severe with the oocyst positive control group, which had a mean lesion score of 0 (significantly lower than the PBS control birds). Birds injected with the 25 µg pcDNA3-SO7′ had a mean lesion score (1.8) significantly less than the pcDNA3 control. All other groups had mean lesion scores >2 and were not significantly different from their respective controls. Figure 2 shows in detail the distribution of lesion severity among birds. The oocyst positive control group was well protected (100% of birds were free of lesions). In contrast, the majority (80%) of birds in the PBS negative control group demonstrated moderate (lesion score of 2.0 to 2.9) to severe (lesion score of 3.0 to 4.0) lesions. In the 12.5 µg pcDNA3-SO7′ group, all of the birds developed lesion scores ≥2.0. However, the majority (50%) of birds injected with the 25 µg dose showed only minor damage to their cecal mucosal tissue, as evidenced by lesion scores <2.0. The 50 µg pcDNA3SO7′ group followed the trend observed for the first set of experiments in which protection decreased with larger doses of pcDNA3-SO7′ (80% of birds developed lesion scores ≥2.0). Some evidence of protection was found in the CheY-SO7′-treated chickens; 40% had lesions <2.0. The corresponding CheY-treated control birds had lesions ≥2.0 in 80% of the birds. For both experiments, variances did not differ significantly between groups. Chicken Weight Gains. Average daily weight gain (grams) in chickens from day of challenge to 7 d following challenge are summarized in Table 1 for each treatment.
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Lesion Scores. In the preliminary experiment, cecal lesions evaluated at 7 d postchallenge (Figure 1A) were
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KOPKO ET AL. TABLE 1. Average daily weight gain (grams) in chickens from day of challenge to 7 d postchallenge during Experiment 11
Group
Number
Treatment
Average daily gain (g) ±SE
1 2 3 4 5 6 7 8
3 3 3 3 3 3 3 3
PBS (1 mL per os) pcDNA3-SO7′ (25 µg i.m. breast) pcDNA3-SO7′(60 µg i.m. breast) pcDNA3-SO7′ (100 µg i.m. breast) pcDNA3 (100 µg i.m. breast) CheY (1 µg s.c.) CheY-SO7′ (1 µg s.c.) Oocyst (2.5 × 103 per os)
16.71 18.09 17.29 18.29 16.33 15.33 13.71 17.62
± ± ± ± ± ± ± ±
0.459 0.504 1.784 1.692 0.585 0.826 1.622 0.882
1 Chickens were treated at 1 wk and 3 wk of age, and were challenged at 5 wk of age with 2.5 × 103 oocysts of Eimeria tenella USDA Strain 80.
DISCUSSION Experiments were conducted to assess the effects of the route of administration and form of a single coccidial antigen (refractile body antigen SO7′) on the resulting immune response of birds. This response was measured using biological indicators of immunological protection against coccidial challenge. Specifically, birds were challenged with virulent parasites, and the rate of growth and degree of protection (if any) from cecal lesions were determined. Other than natural infections induced by oocysts administered per os, intramuscular injection with the 25-µg
dose of plasmid DNA containing the SO7′ insert under the control of a viral promoter induced the greatest level of protection against challenge with E. tenella infection. In both experiments, lesion scores were reduced to 0 to 1.5 in more than 50% of the chickens. In comparison, 90% of the control chickens showed a moderate to severe degree of infection (lesion score range 2.0 to 4.0). Decreased damage to the cecal mucosa in chickens injected with 25 µg pcDNA3-SO7′ suggests the involvement of an immune effector mechanism that may have resulted in attrition or inhibition of development of early stages of the parasite life cycle such as sporozoites or first-generation merogonic stage. The inability of this plasmid DNA to significantly decrease lesion scores in 100% of the vaccinated subjects may be attributed to variability in expression of the recombinant antigen among birds in a single experiment and between experiments. Chickens injected with 12.5 µg pcDNA3-SO7′ and high doses of pcDNA3-SO7′ (50, 60, or 100 µg) demonstrated severe lesions comparable with those of the pcDNA3 control (100 µg). Previous immunization studies (H. ProfousJulchelka and M. Hozza, 1993, Merck & Co., Inc., PO Box 2000, R8OY-265, Rahway, NJ 07065, personal communication) have found that multiple injections of 100 µg of plasmid DNA (RSV expression vector containing SO7′ gene) did not elicit protection against lesion scores in challenge infections. A possible explanation for the lack
TABLE 2. Average daily weight gain (grams) in chickens from day of challenge to 7 d postchallenge during Experiment 21
Group
Number
Treatment
Average daily gain (g) ±SE
1 2 3 4 5 6 7 8
8 8 10 9 7 7 7 10
PBS (1 mL per os) pcDNA3-SO7′ (12.5 µg i.m. breast) pcDNA3-SO7′ (25 µg i.m. breast) pcDNA3-SO7′ (50 µg i.m. breast) pcDNA3 (50 µg i.m. breast) CheY (1 µg s.c.) CheY-SO7′ (1 µg s.c.) Oocysts (2.5 × 103 per os)
11.57 10.95 13.48 12.90 9.97 13.53 13.98 18.97
± ± ± ± ± ± ± ±
0.979 1.307 0.8152 1.200 1.302 1.041 1.003 0.8683
1 Chickens were treated at 1 wk and 3 wk of age, and were challenged at 5 wk of age with 2.5 × 103 oocysts of Eimeria tenella USDA Strain 80. 2 Significantly different from the pcDNA3 control birds (Group 5) at P < 0.05. 3 Significantly different from the PBS control birds (Group 1) and all other treatment groups at P < 0.05.
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The average daily weight gain was greatest in birds inoculated with 25- and 100-µg doses of pcDNA3-SO7′; however, these birds did not demonstrate rates of growth that were significantly greater than that of the pcDNA3 control group. The small sample size resulted in no significant differences between any groups in the first experiment. In the second experiment (Table 2), birds treated with oocysts or the 25-µg dose of pcDNA3-SO7′ showed significantly higher average daily weight gains than did the controls (PBS and pcDNA3, respectively). The average daily weight gain of birds injected with recombinant antigen (CheY-SO7′) was not significantly higher than the CheY-negative control group.
IMMUNIZING STRATEGIES USING A REFRACTILE BODY ANTIGEN
means of immunizing chickens against challenge infections with E. tenella. However, our study demonstrated that a carefully titrated dose of a refractile body antigen delivered to chickens in a DNA-based vaccine construct was able to elicit an immune response that provided significant biological protection (as measured by lesion score reduction and maintained high rate of growth) against virulent E. tenella challenge. This result is the first demonstration of naked DNA vaccination that works in chickens against cecal coccidiosis. These results provide encouragement that cloned coccidial antigens, singly or more likely in combination, may be included in a commercially viable, subunit vaccine against avian coccidiosis (Jenkins, 1998; Vermeulen, 1998). The lack of efficacy of the previous attempts at vaccination using recombinant antigens may be more related to the form of the antigen and its route of delivery to the birds than to the intrinsic immunogenicity of these recombinant coccidial proteins. The DNA vaccination may be preferable to proteinaceous antigens because of the relative ease of production, administration, and storage compared with recombinant proteins.
ACKNOWLEDGMENTS We thank Harry Danforth for providing us with the cell line that secretes mAb 1209; Helen Profous-Juchelka, Julie Cobean, Susan Slack, Krystina Strickler, Muhammad Tahir, Cindy Kopko, and Sunny Mann for technical assistance; and Steve Gismondi and William Sears for their help with the statistical analyses. We also thank Aggie Fernando, Ramon Carreno, and Krystina Strickler for their criticisms of earlier drafts of this manuscript. This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada (106453-98) and the Ontario Ministry of Agriculture, Food and Rural Affairs Research Program to John R. Barta.
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of protection with the high doses of pcDNA3-SO7′ may be derived from studies on DNA vaccines in other systems. Mice immunized with different doses of a Plasmodium yoelli circumsporozoite protein DNA-based vaccine exhibited a dose-related response in which antibody levels increased with greater amounts of injected DNA (Gramzinski et al., 1997). By analogy, doses of pcDNA3SO7′ >25 µg may be inducing primarily an antibody response that has been shown to play only a minor role in protection against coccidial infection (Rose et al., 1984; Augustine and Danforth, 1986). The inability to protect chickens with the 12.5-µg dose of pcDNA3-SO7′ may be due to expression of the antigen at levels insufficient to elicit any immune response and would explain why lesions and weight gains were similar to those of the PBS controls. Most chickens treated with the recombinant antigen (CheY-SO7′) were not protected against cecal lesions. Past studies on immunization of chickens with CheY-SO7′ (Crane et al., 1991) showed that a single dose, without adjuvant, protected against severe coccidiosis induced by infection with E. tenella. In addition, chickens treated with CheY-SO7′ exhibited protection against challenge with heterologous species (Eimeria acervulina, Eimeria maxima, and Eimeria necatrix). However, the mean lesion score was never reduced below 1.5, indicating that immunization with the fusion protein CheY-SO7′ only elicited a low level of protection. In our studies, the dose of CheY-SO7′ (1 µg) was comparable with that used by Crane et al. (1991). According to their reports, increasing the dose of CheY-SO7′ would not have improved the observed protection in this study. Differences in the type of chicken used in the present experiment (White Leghorn layers vs production broilers) or antigen delivery (with or without adjuvant) may explain the differences in response to the same recombinant antigen observed in the present experiments and by Crane et al. (1991). Another biological measure used to assess protection against coccidial infection in chickens is weight gain following challenge. Protection against coccidial challenge is indicated by a high average daily weight gain as seen with the oocyst group, which was immunized multiple times with the parasite throughout the experiment. The negative control group (PBS) had a significantly lower rate of growth than the oocyst-positive control in the second immunization trials. The only experimental group to exhibit weight gain significantly greater than their negative control group was the 25 µg pcDNA3-SO7′ treatment group in the second set of experiments. Again, treatment with the 25 µg pcDNA3-SO7′ induced an intermediate response between pcDNA3 (negative control) and oocyst (positive control) treatments, as was seen with lesion scores. The only study with a coccidial subunit vaccine to demonstrate some degree of protection against both weight loss and lesion scores in birds was one using a fusion protein containing a merozoite surface antigen (MZ250) (Jenkins et al., 1991). Overall, these immunization trials demonstrate that the viable parasite form (oocyst) is still the most effective
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