Immune Responses and Resistance to Eimeria acervulina of Chickens Divergently Selected for Antibody Responses to Sheep Red Blood Cells

Immune Responses and Resistance to Eimeria acervulina of Chickens Divergently Selected for Antibody Responses to Sheep Red Blood Cells H. K. Parmentier,*,1 S. Yousif Abuzeid,* G. De Vries Reilingh,* M. G. B. Nieuwland,* and E. A. M. Graat† *Health and Reproduction Group, and †Quantitative Veterinary Epidemiology Group, Wageningen Institute of Animal Sciences, Wageningen University, PO Box 338, 6700 AH Wageningen, The Netherlands put found in the H line as compared to the C and L lines. After secondary infection, significantly higher fecal oocyst output was found in the C line. Significantly higher Ab response after primary and secondary infection were found in the H and C lines as compared to the L line. No line differences were found for cellular immune responses to E. acervulina oocyst antigen. These observations imply that selection for enhanced humoral immunity to SRBC did not result in enhanced resistance to E. acervulina in terms of fecal oocyst output. However, the H line might expel E. acervulina more rapidly than the other two lines. The absence of line differences in resistance to Eimeria is discussed with respect to the role of the humoral immune response.

(Key words: chicken, selection, sheep red blood cells, immune response, coccidiosis) 2001 Poultry Science 80:894–900

Prohibition of antibiotics, as well as the risk of resistance to anticoccidials, indicates that a search for alternative ways to maintain the health of poultry stock may be necessary. (In)direct selection for general enhanced disease resistance or general decreased susceptibility with selection for an immune response induced by a harmless (poly-)antigen such as SRBC has long been considered as an attractive supplement of disease control in poultry. Several chicken lines selected for high antibody (Ab) responses to SRBC have been shown to be more resistant to Eimeria spp., but, in contrast, appeared more susceptible to Escherichia coli and Staphylococcus aureus infections (Gross et al., 1980; Dunnington et al., 1986). Low correlations between immune traits indicated it may be possible to select for or improve separate components of the immune system (Cheng and Lamont, 1988, 1990) by multitrait selection on various immune responses. Studies

with broilers selected for high early Ab production to E. coli and Newcastle disease virus vaccines indicate that it may be possible to select for enhanced maturation of the complete immune response at a young age and to obtain simultaneously enhanced cellular, humoral, and phagocytic responses against unrelated antigens or pathogens, the latter being representative of separate types of immune responses (Pitcovsky et al., 1987a,b; Heller et al., 1992). Two layer lines were divergently selected for high (H line) or low (L line) Ab responses to SRBC injected i.m. at 37 d of age (Van der Zijpp and Nieuwland, 1986). A randombred control (C) line resembling the genetic pool of the original parental stock (Pinard et al., 1993b) was also maintained. The present lines differ in humoral responses to various unrelated T cell-dependent antigens and vaccines (Kreukniet et al., 1992; Parmentier et al., 1993, 1996, 1998). We studied the effect of divergent selection on Ab responses to SRBC on the resistance of the

2001 Poultry Science Association, Inc. Received for publication September 18, 2000. Accepted for publication February 5, 2001. 1 To whom correspondence should be addressed: henk.parmentier@ genr.vh.wau.nl.

Abbreviation Key: Ab = antibody; ConA = concanavalin A; C = control line; H = high antibody line; L = low antibody line; LST = lymphocyte stimulation test; OPG = oocysts per gram; PBL = peripheral blood leukocytes; SI = stimulation indices.

INTRODUCTION

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ABSTRACT Resistance to Eimeria acervulina was measured in two lines of chickens that had been divergently selected for high (H line) or low (L line) antibody (Ab) responses to SRBC, and in a randombred control (C) line originating from the same parental stock. Fecal oocyst output of cocks from the three lines from the 17th generation was estimated after primary and secondary infection with 2 × 105 oocysts. In addition, Ab responses to E. acervulina oocyst antigen and cellular immune responses in vitro to E. acervulina antigen were measured after primary and secondary infection with E. acervulina. No significant line differences were found with respect to fecal oocyst output after primary infection. Only at the end of the primary infection period, i.e., Day 15 postprimary infection, was a significantly lower fecal oocyst out-

SELECTION LINES AND RESISTANCE TO EIMERIA

three lines to E. acervulina, which next to E. tenella is a very frequently diagnosed Eimeria strain in poultry in the Netherlands (Voeten, 1987; Graat, 1996). Resistance of adult cocks of the 17th generation was measured. In addition, humoral and cellular immune responses toward E. acervulina were studied in the three lines after primary and secondary infections.

MATERIALS AND METHODS Birds and Housing

Parasite Strain Eimeria acervulina (strain originating from Houghton Poultry Institute) was maintained and kindly provided by the Dutch Animal Health Service.2

Parasite Antigen E. acervulina oocyst antigen was prepared as follows. Feces from birds that had been infected with E. acervulina 5 d earlier were mixed with saturated NaCl solution, sieved, and centrifuged three times at 225 × g. After repeated washing with tap water and centrifugation at 2,700 × g, 400 × g, 200 × g, and 100 × g, respectively, oocysts were sporulated after 24 to 48 h at 29 C in tap water with a continuous supply of fresh air. After centrifugation at 100 × g, oocysts (90% sporulated) were either frozen until use or disrupted by shaking with 0.5 mm glass beads. Protein content was determined in supernatant, which was centrifuged at 500 × g and subsequently at 14,000 rpm in an Eppendorf centrifuge,3 with a Lambda 1 UV/ vis spectrophotometer4 according to Bradford (1976).

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Deventer, The Netherlands. Eppendorf, 2000 Hamburg 63, Germany. 4 Perkin Elmer, Norwalk, CT 06859. 5 Merck, Sharpe & Dohme, 2003 PC Haarlem, The Netherlands. 6 Fisher Scientific bv, 5213 AS ‘s-Hertogenbosch, The Netherlands. 7 Nordic, 5000 AA Tilburg, The Netherlands. 8 Labsystems, FIN-00881 Helsinki, Finland. 9 Amersham Pharmacia Biotech Benelux, 4700 BJ Roosendaal, The Netherlands. 10 ICN Biomedicals Inc., Aurora, Ohio 44202. 3

Experimental Design Ten cocks, 5 mo of age, of each line were infected directly in the crop with 2 × 105 sporulated E. acervulina oocysts in 1 mL tap water with a 1 mL syringe at Day 0. On Days 5, 6, 7, 8, 12, 15, and 20 after infection all feces were collected from each bird and thoroughly mixed. Five-gram feces samples from each bird were examined in duplicate for oocysts with a McMaster5 counting chamber according to Long and Rowell (1975). Ten cocks of each line were not infected and received only 1 mL of tap water. Thirty-five days after initial primary inocula, only the infected cocks received a second inoculum with 2 × 105 sporulated oocysts. All feces were collected from each bird at Days 5, 9, and 14 postsecondary infection. The number of oocysts in the inoculum was determined with a Fuchs-Rosenthal hemocytometer.6

Humoral Immune Response to Eimeria acervulina Total Ab titers to E. acervulina oocyst antigen were determined by ELISA in serum from all 60 (infected and noninfected) cocks 1 d before, and on 4, 17, 21, and 28 d after primary infection, at the day of secondary infection, and 7, 14, and 21 d thereafter. Briefly, 96-well plates were coated with 10 µg E. acervulina antigen/mL. After subsequent washing with PBS and 0.05% Tween, the plates were incubated with serial dilutions of serum. Binding of Ab to E. acervulina oocyst antigen was detected using 1:20,000 diluted RACh/IgGH+L/PO.7 After washing, tetramethylbenzidine and 0.05% H2O2 were added, and the plates were incubated for 10 min at room temperature. The reaction was stopped with 2.5 N H2O4. Extinctions were measured with a Multiskan8 at a wavelength of 450 nm. Titers were expressed as the log2 values of the highest dilution giving a positive reaction. Positivity was derived from the extinction values of a standard positive serum present on every microtiter plate.

In Vitro Lymphoproliferation to Eimeria acervulina Antigen E. acervulina oocyst antigen-specific in vitro cellular immunity was determined by a lymphocyte stimulation test (LST). At 28 d postprimary infection, and at 28 d postsecondary infection, peripheral blood leukocytes (PBL) were obtained from heparinized blood from all birds using Ficoll density gradient centrifugation. In brief, 1 mL blood was layered on 0.5 mL Ficoll-Paque9 and centrifuged for 2 min at 14,000 × g in an Eppendorf centrifuge, after which PBL were collected from the interphase. PBL were tested for proliferation at a final concentration of 5 × 106/mL with addition of 5 µg/mL concanavalin A (ConA), 5 µg/ mL E. acervulina oocyst antigen, or 10 µg/mL oocyst antigen in RPMI-tissue culture medium supplemented with 2 mM L-glutamine10 (cat. no. 1060120), 1% nonimmune chicken plasma, 5 × 10−5 M 2-mercaptoethanol, 100 µg/ mL streptomycin, and 100 IU/mL penicillin in 96-well

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Cocks originated from an ISA Warren cross (medium heavy layers). This cross has been selected in the past for H and L primary Ab responses at Day 5 after primary i.m. immunization with 1 mL of 25% packed SRBC at 37 d of age. Additionally, a randombred C line originating from the same parental stock was maintained (Van der Zijpp and Nieuwland, 1986). Birds from the 17th generation were used. They were housed individually in brooder batteries with free access to water and to feed without anticoccidials (152 g/kg CP, 2,817 kcal/kg ME). The birds were vaccinated against Marek’s disease, infectious bronchitis, and infectious bursal disease at hatch and 2 and 15 d of age, respectively.

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FIGURE 1. Time course of the mean fecal oocyst per gram feces (OPG) of cocks from the high (+), control (䉭), and low (䊊) antibody lines after primary and secondary (arrow) infections with 200,000 sporulated Eimeria acervulina oocysts.

Statistical Analysis Differences between fecal oocyst counts and ln-transformed fecal oocyst counts after primary and secondary infections were tested by a two-way ANOVA for the effect of line and time and for their interactions by using the repeated measurement procedure in which cock was nested within line. Also, a one-way ANOVA was used to determine differences in fecal oocyst output between lines at each day of sampling after infection. Primary and secondary serum Ab titers to E. acervulina were analyzed by a three-way ANOVA for the effect of line, infection, and time and their interactions by using the repeated measurement procedure. In vitro lymphoproliferation at Days 28 postprimary and secondary infections, were analyzed by a two-way ANOVA for the effect of line and infection and their interactions. All analyses were according to SAS software (1985) procedures. Mean differences between lines and between infection treatments were tested with Bonferroni’s test.

RESULTS Fecal Oocyst Counts Figure 1 shows the pattern of fecal oocyst excretion [oocysts per gram (OPG) feces nontransformed data] of

11

ICN Pharmaceuticals Inc., Costa Mesa, CA 92626. Beckman Instruments, 3641 RP Mijdrecht, The Netherlands.

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Primary and Secondary Humoral Responses to Eimeria acervulina Primary Response. The kinetics of anti-E. acervulina Ab titers in serum during 5 wk after primary infection are shown in Figure 2. The highest titers were found at Days 17 and 21 postinfection. All birds infected with E. acervulina mounted low Ab responses to E. acervulina oocyst antigen, which were, however, significantly higher than titers of uninfected birds (P < 0.05, three-way ANOVA, Table 2). There was a significant line effect; H line birds mounted significantly higher titers than C line birds, and C line birds had higher titers than L line birds (P < 0.05, Table 2). A line-by-time interaction was found, but no interaction between line and infection was observed. Secondary Response. The kinetics of anti-E. acervulina Ab titers during the 3 wk after secondary infection are shown in Figure 2. Ab titers were highest at 14 d after secondary infection in all three lines (Figure 2). The H and C lines mounted significantly higher Ab titers to E. acervulina than the L line (P < 0.05, three-way ANOVA, Table 3). Similarly as during the primary response, no significant line × infection interaction was found during the secondary Ab response.

In Vitro Responses to ConA and Eimeria acervulina Antigen Cellular responsiveness in vitro to the mitogen ConA and two doses of E. acervulina oocyst antigen at Day 28 after primary and secondary infections, with 2 × 105 sporulated E. acervulina oocysts, are shown in Table 3. The mitogenic responses (SI) to ConA at both sampling times were significantly affected by line, i.e., a significantly higher mitogenic response was found with PBL from the L line at both sampling days (two-way ANOVA). A significantly enhanced specific response to E. acervulina antigen was found only after secondary infection with a challenge dose of 10 µg/mL. No line-by-infection interactions were found with respect to the mitogenic or antigen specific responses at either time.

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flat-bottomed plates. After incubation for 48 h (ConA) or 72 h (E. acervulina) at 41 C and 5% CO2, the cultures, set up in triplicate, were pulsed for 18 h with 0.5 µCi methyl[3H]-thymidine.11 [3H]-Thymidine uptake was determined with a Beckman β-scintillation counter.12 Results were expressed as mean stimulation indices (SI). The SI were calculated as SI = counts per min (cpm) in antigen- or ConA-stimulated cultures/cpm in unstimulated cultures.

the infected cocks of all three lines. Overall, after primary infection, no significant line differences were found (twoway ANOVA, repeated measures, Table 1). Only at the end of the primary infection (Day 15) were significantly fewer oocysts found in the feces of the H line that in the L line or C line (one-way ANOVA, P < 0.05). At that day, the mean OPG were 51, 149, and 305 for H, C, and L lines, respectively. Also at Day 12, fewer oocysts were found in the feces of the H line (mean OPG of 777 vs. an OPG of 1,424 in C line feces and an OPG of 2,854 in L line feces), but this difference was not significant. No oocysts were found in the noninfected birds. After secondary infection, significantly more Eimeria oocysts were found in feces of the C line cocks (P < 0.05) compared to the H and L line cocks (Table 1).

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SELECTION LINES AND RESISTANCE TO EIMERIA TABLE 1. (Ln) Oocysts per gram of feces from high (H), control (C), and low (L) antibody line cocks after primary [(ln) OPGI], and secondary oral [(ln) OPGII] infections with 2 × 105 sporulated E. acervulina oocysts Line1

OPGI2

ln OPGI2

OPGII2

ln OPGII2

H C L

5,961,061 2,988,208 4,389,777

12.67 12.53 12.72

322,053a 2,254,430b 178,833a

SEM

1,069,273

10.15 11.35 10.84 0.502

Main effects Line Time Line × time

NS *** NS

0.363 NS *** NS

604,484 H, L < C* * *

NS *** NS

a,b

Values in columns with different superscripts differ significantly. 10 cocks per line. 2 Least square means of oocysts per gram feces (OPGI) of the entire observation period after primary oral infection: Days 5 to 15 and Days 5 to 14 after secondary oral infection (OPGII). *P < 0.05. ***P < 0.001. 1

We studied the effect of divergent selection of layers for Ab responses to SRBC on the resistance to E. acervulina as determined by fecal oocyst output. Other parameters of infection, such as BW, feed conversion, and intestinal inflammation, were not included in this study. The rapid emergence of drug-resistant Eimeria strains (Chapman, 1993) may intensify the search for alternatives to chemotherapeutics. Enhanced immune-based natural disease resistance of food animals may be an attractive supplement to preventive medication in general. Genetic variation in resistance of chicken breeds has been reported (Jeffers and Shirley, 1982), as well as various other factors that influence resistance and susceptibility of flocks to Eimeria spp. (Bumstead and Millard, 1992). Layers as well as broilers (Gross et al., 1980; Dunnington et al., 1986) have been selected for their humoral immune responses to nonpathogenic multiantigens that exhibit higher resistance

FIGURE 2. Time course of the mean systemic antibody titers to Eimeria acervulina sporulated oocyst antigen of infected (solid lines, solid symbols) and noninfected (control) cocks (dashed lines, open symbols) from the high (+), control (䉭), and low (䊊) antibody lines after primary and secondary (arrow) infections with 200,000 sporulated E. acervulina oocysts.

in terms of growth rates and lesion scores to Eimeria spp. in the high Ab producing lines and lower resistance to Eimeria spp. in the low Ab producing lines. The present lines, selected for Ab responses to SRBC, showed no difference in resistance to E. acervulina as measured by fecal oocyst output. This study used cocks from the 17th generation, 5 mo of age, and infected twice with one dose of oocysts instead of a trickle infection as might occur in practice. Similar results were found with 9-mo-old hens of the 13th generation that were infected with 105 of E. acervulina oocysts (data not shown). Also in these birds, a line difference was found only at Day 13 postinfection, i.e., at the end of the infection period when OPG are already low (unpublished data). Progressive divergent selection on the Ab response apparently did not result in a change in the resistance to E. acervulina between the lines. Different infection doses, sexes, and ages, also might not affect resistance of the three lines to E. acervulina. In the present study, a secondary infection did not result in different oocyst outputs between the H and L lines. On the contrary, the H and L lines had less oocyst output than the C line, as if both lines had been selected for protective immunity, although based on different mechanisms. It remains to be studied whether birds of much younger ages and infected with various other doses respond similarly. A clear difference between the lines in the Ab response against E. acervulina was found in the primary and the secondary responses. Thus, divergent selection on Ab responses to SRBC also resulted in differences in Ab responses to E. acervulina, but these Ab levels may have no causal relation with resistance to E. acervulina in terms of termination of infection or expulsion of the parasite from the intestinal tract. With respect to the kinetics of oocyst release, the present L line terminated the infection in a similar fashion to the H and C lines, but no Ab were found in the L line during primary infection. On the other hand, the L line mounted Ab after secondary infection, indicating that its incapacity to mount Ab is limited to primary responses. No clear effect of higher Ab titers in

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DISCUSSION

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PARMENTIER ET AL. TABLE 2. Serum antibody titers against Eimeria acervulina oocyst antigen from high (H), control (C), and low (L) line chickens after primary oral infection at 5 mo of age with 2 × 105 sporulated E. acervulina oocysts Group1 Line

Treatment

H 1. H 2. C 1. C 2. L 1. L 2. SEM Main effects5 Line Infection Line × infection Time Line × time Infection × time Line × infection × time

E. acervulina tap water E. acervulina tap water E. acervulina tap water

Primary titer2

Secondary titer2

2.04a 1.38b 1.63b 0.96c 1.01c 0.690c 0.123

9.13a 1.97c 9.28a 1.66c 7.16b 0.98c 0.478

*** H > C > L *** E. acervulina > water NS *** * *** NS

** H ≥ C > L *** E. acervulina > water NS *** ** *** *

a,b

the H and C lines, as compared to the L line, on resistance in terms of oocyst output was found. However, when chickens, irrespective of line, were grouped on the basis of their cumulative oocyst output after secondary infection, the secondary infection was less pronounced in those birds that had significantly higher Ab titers against E. acervulina during primary infection (data not shown),

which suggests that Ab might play a role in the prevention of secondary infection with E. acervulina. The three lines differed in Ab titers to E. acervulina antigen as determined by ELISA, but the mechanisms underlying lower titers, i.e., different recognition of antigenic fragments of E. acervulina or similar recognition but lower production of Ab, are unknown. Western blot

TABLE 3. Cellular responsiveness in vitro of high (H), control (C), and low (L) line cocks to concanavalin A (ConA) and Eimeria acervulina antigen after primary and secondary oral infection with 2 × 105 sporulated E. acervulina oocysts at 5 mo of age Stimulation index4 ConA3

Group 1 Line H H C C L L SEM Main effects Line

2

Treatment 1 2 1 2 1 2

Infection Line × infection a,b

I

Eimeria [5] II

b

a

Eimeria [10]

I

II

I

II

279 145a 316b 256a 318b 317b 43.5

178 247ab 264ab 234ab 337b 429b 46.2

2.66 1.27 1.99 1.13 1.79 1.15 0.89

1.30 1.87 1.48 1.15 1.41 1.02 0.26

1.70 0.98 1.76 1.07 1.21 1.07 0.40

1.58 0.88 1.63 0.93 2.21 0.77 0.53

* H, C < L NS NS

** H, C < L

NS

NS

NS

NS

NS NS

NS NS

NS NS

1>2* NS

Different superscripts between line (column) denote significant differences. 10 cocks per group (line and treatment). 2 Treatment 1 = taken orally 2 × 105 sporulated E. acervulina oocysts; Treatment 2 = tap water taken orally. 3 I = lymphocyte stimulation test at 28 d postprimary infection; II = at 28 d postsecondary infection. 4 Least square means of the stimulation indices, background of unstimulated cultures averaged 200 cpm. Eimeria [5] = 5 µg/mL E. acervulina oocyst antigen, Eimeria [10] = 10 µg/mL E. acervulina antigen. *P < 0.05. **P < 0.01. 1

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Values in columns with different superscripts differ significantly. 10 cocks per group (line and treatment). 2 Least square means of the entire observation period. *P < 0.05. **P < 0.01. ***P < 0.001. 1

SELECTION LINES AND RESISTANCE TO EIMERIA

REFERENCES Bradford, M. M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing

the principle of protein-dye binding. Anal. Biochem. 72:248–254. Bumstead, N., and B. J. Millard, 1992. Variation in susceptibility of inbred lines of chickens to seven species of Eimeria. Parasitology 104:407–413. Chapman, H. D. 1993. Resistance to anticoccidial drugs in fowl. Parasitol. Today 9:159–162. Cheng, S., and S. J. Lamont, 1988. Genetic analysis of immunocompetence measures in a White Leghorn chicken line. Poultry Sci. 67:989–995. Cheng, S., and S. J. Lamont, 1990. Selection for general immunocompetence in chickens. Pages 58–61 in: Proceedings 4th World Congress on Genetics Applied to Livestock Production, W. G. Hill, R. Thompson, and J. A. Woolliams, ed. Vol. XVI, International Communications of World Congress on Genetic Applied Livestock Production, Edinburgh, Scotland. Dunnington, E. A., A. Martin, W. E. Briles, R. W. Briles, and P. B. Siegel, 1986. Resistance to Marek’s disease in chicken selected for high and low antibody response to lower case “s” sheep red blood cells. Arch. Geflu¨gelk. 50:94–96. Graat, E.A.M., 1996. Epidemiology of Eimeria acervulina infections in broilers: An integrated approach. Ph.D. Thesis. Wageningen University, Wageningen, The Netherlands. Gross, W. G., P. B. Siegel, R. W. Hall, C. H. Domermuth, and R. T. DuBoise, 1980. Production and persistence of antibodies in chickens to sheep erythrocytes. 2. Resistance to infectious diseases. Poultry Sci. 59:205–210. Heller, E. D., G. Leitner, A. Friedman, Z. Uni, M. Gutman, and A. Cahaner, 1992. Immunological parameters in meat-type chicken lines divergently selected by antibody response to Escherichia coli vaccination. Vet. Immunol. Immunopathol. 34:159–172. Jeffers, T. K., and M. W. Shirley, 1982. Genetics, specific and intraspecific variation. Pages 63–100 in: The Biology of the Coccidia. P. L. Long, ed. University Park Press, Baltimore, MD. Kreukniet, M. B., S.H.M. Jeurissen, M.G.B. Nieuwland, N. Gianotten, P. Joling, and H. K. Parmentier, 1996. The B cell compartment of two chicken lines divergently selected for antibody production: differences in structure and function. Vet. Immunol. Immunopathol. 51:157–171. Kreukniet, M. B., N. Gianotten, M.G.B. Nieuwland, and H. K. Parmentier, 1994. In vitro T cell activity in two chicken lines divergently selected for antibody response to sheep erythrocytes. Poultry Sci. 73:336–340. Kreukniet, M. B., A. J. van der Zijpp, and M.G.B. Nieuwland, 1992. Effects of route of immunization, adjuvant and unrelated antigens on the humoral immune response in lines of chickens selected for antibody production against sheep erythrocytes. Vet. Immunol. Immunopathol. 33:115–127. Lillehoj, H. S., and J. M. Trout, 1996. Avian gut-associated lymphoid tissues and intestinal immune responses to Eimeria parasites. Clin. Microbiol. Rev. 9:349–360. Long, P. L., and J. G. Rowell, 1975. Sampling broiler house litter for coccidial oocysts. Br. Poult. Sci. 16:583–592. Parmentier, H. K., M. B. Kreukniet, B. Goeree, T. F. Davison, S.H.M. Jeurissen, E.G.M. Harmsen, and M.G.B. Nieuwland, 1995. Differences in distribution of lymphocyte antigens in chicken lines divergently selected for antibody responses to sheep red blood cells. Vet. Immunol. Immunopathol. 48:155–168. Parmentier, H. K., M.G.B. Nieuwland, E. Rijke, and J. W. Schrama, 1996. Divergent antibody responses to vaccines and divergent body weights of chicken lines selected for high and low humoral responsiveness to sheep red blood cells. Avian Dis. 40:634–644. Parmentier, H. K., J. W. Schrama, F. Meijer, and M.G.B. Nieuwland, 1993. Cutaneous hypersensitivity responses in chickens divergently selected for antibody responses to sheep red blood cells. Poultry Sci. 72:1679–1692.

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analyses with the same immune sera as used for ELISA from all three lines showed significant staining of a fragment of E. acervulina oocyst antigen < 10 kDa, but no significant line differences in staining intensities were found (data not shown). The current lines, thus, may not differ in their capacity to mount specific idiotypes but may, rather, differ in their capacity to raise Ab levels to E. acervulina and other antigens. Previously, Kreukniet et al. (1996) and Parmentier et al. (1995) found large differences between the current selection lines in the numbers of B cells and B-cell areas in the peripheral blood and spleen. Whether sporulated oocyst antigen is appropriate and relevant to measure immune responses to E. acervulina infected chickens is questionable; however, oocyst antigen is accessible; it was bound by Ab in ELISA, and it stimulated lymphocytes in vitro. In vitro cellular immune responses against E. acervulina did not differ between the lines. Only the in vitro mitogen response was significantly higher in the L line as reported previously (Kreukniet et al., 1994). Higher levels of CD8+ cells and TCR-1 (γ/∆) cells are present in the L line (Parmentier et al., 1995). As yet it is unknown whether the difference in mitogen response is related to the different cellular compositions between the lines. With the exception of a marginal difference, selection for enhanced Ab responses of layers to SRBC after 17 generations did not result in improvement of resistance to E. acervulina under the present experimental conditions. In the present lines we could also not find line differences with cellular immunity in vitro, with the exception of the mitogenic response. These results are not contradictory to a prominent role of the cellular immune response in the resistance to Eimeria. Resistance to Eimeria appears mainly cell-mediated, but the presence of serum Ab and IgA during infection does not exclude a complementary role of Ab in resistance to Eimeria (Lillehoj and Trout, 1996; Trout and Lillehoj, 1996; Zigterman et al., 1993). A lack of difference in resistant to E. acervulina does not imply that these lines should respond similarly to other Eimeria spp. Inverse relationships between susceptibility to different Eimeria species have been described (Bumstead and Millard, 1992). However, the present results resemble findings of Pinard et al. (1993a) who reported that no enhanced resistance to Marek’s virus was found in the H line, but a decreased resistance to the virus was found in the L line. Earlier results with these lines indicated that enhancement Ab responses did not lead to enhancement of Ab responses to other T-celldependent antigens of the H line as compared to the C line (Parmentier et al., 1994). Thus, the present results do not support the concept of enhancement of nonantigenspecific disease resistance by divergent selection on the humoral immune response. The correspondence in resistance and in the cellular responses of the present lines suggests that selection on cellular responsiveness might add to enhancement of resistance to coccidiosis.

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