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Delayed wattle reactions in Eimeria tenella infected chickens
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. I0, pp. 387-394, 1986. 0145-305X86 $3.00 + .00 Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.
DELAYED WATILE REACTIONS IN EIMERIA TENk~,TA INFECTED CHICKENS
Robert L. Taylor, Jr., Richard G. Strout, Robert A. Clare and Paul A. Aeed Department of Animal and Nutritional Sciences University of New Hampshire Durham, New Hampshire USA 03824
ABSTRACT
Wattle reactions to a n ~ t e n e l l a antigen and phytohemagglutinin (PHA) were studied in chickens infected with E. tenella. Two trials were conducted using a total of 224 chickens. Four days after infection with one dose of i0,000 sporulated oocysts, the increase in wattle thickness in response to E. tenella antigen was significantly greater than that of uninfected controls. This significant response persisted through day i0 post infectio~ Wattle responses to PHA 3 days after infection were significantly greater than for uninfected controls. Significant differences in response to PHA were maintained throughout the experiment except on day 6. An increased response to PHA from days 7 to 13 post infection was attributed to the lower parasite burden at that time.
INTRODUCTION Cellular immunity in chickens has been assessed by delayed wattle reactions (DWR) (i). In this technique, sensitized birds are injected intradermally in the wattle with the sensitizing antigen and the resulting swelling in the injected wattle is then used to determine the degree of response. Such tests are performed easily and have been correlated with other tests of T cell function such as spleen cell migration inhibition and lymphocyte blastogenic responses (i). Antigens t e s t e d in this s y s t e m i n c l u d e t u b e r c u l i n (2-5), dinitrophenylated chicken serum albumin (3), diphtheria toxin (1,6), human g a m m a globulin (7,8), and bovine serum albumin (7,9). Disease organisms tested include S a l m o n e l l a adelaide (3), Marek's disease virus (i0) and coccidia (11-14). Phytohemagglutinin (PHA), a mitogenic lectin extracted from v~laaris, has also been used in wattle tests. This reaction proceeds without prior sensitization and is mediated by T cells (15,16). The first cells to 387
388
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infiltrate the wattle were heterophils and mononuclear cells in as little as 30 min. followed by basophils and eosinophils 24 hours after injection. Granulocyte numbers declined by 48 hours while lymphocytes increased (17). Multinucleated giant cells were also observed (18) in the region of infiltration. Rose and Hesketh (1984) reported that infection with Eimeria tenella depressed the T lymphocyte blastogenic response to PHA (19). In contrast, the T cell response to E. tenella antigen increased during the course of parasitism. Giambrone and coworkers (20) found a high correlation between innate resistance and DWR in lines selected for susceptibility and resistance to coccidiosis. Their tests were performed 2 to 6 weeks after initial exposure. The DWR to coccidia antigens during the time immediately following infection has not been investigated. This paper reports the wattle responses to an antigen from E. tenella and to PHA in birds during the first 13 days after infection with E. tenella.
MATERIAL S AhD M E ~ O D S Animals: Straight run Single Comb White Leghorns were used throughout this study. Birds were maintained according to standard management practices at the University of New Hampshire Poultry Research Farn~ The chicks were raised coccidia-free in heated metal batteries with free access to unmedicated feed and water. Vaccinations against Marek's disease and Newcastle-bronchitis were administered during the growing period. Coccidia cultures: The Lilly 65 strain of Eimeria tenella (Eli Lilly, Greenfield, IN9 was maintained in our laboratory. Viability and pathogenicity were preserved by serial passage at regular intervals. Suspensions of counted oocysts were administered to each chicken orally using an inoculation tube and syringe. ~Jl~q~: Sterile oocysts were suspended in 20 m~ sterile phosphate buffered saline (PBS, pH 7.2) at a concentration of 10 v oocysts/ml. The oocyst suspension was twice disrupted at 10,000 PSI in a sterile French Press (Fred S. Carver, Inc., Summit, NJ) and subjected to two freeze-thaw cycles. The antigen preparation was kept frozen until use. lli~: A stock solution containing 1 mg/ml of phytohemagglutinin (PHA-P, Difco Laboratories, Detroit, MI) was prepared in sterile PBS (pH 7.2). Wattle test: TWo trials, each consisting of 112 animals, gave a total of 224 chickens. A t six weeks of age, birds were randomly divided into 14 groups of 8 birds each. Group 1 (Day 0) was designated the uninfected control. The remaining 13 groups received a single inoculum of 10,000 sporulated oocysts of Eimeria ~ On each of 14 consecutive days, birds from one group were tested for their wattle reaction to either E. tenella antigen or PHA. The wattle test was conducted by measuring the thickness of each wattle with a micrometer. After measurement, either 0~I ml of the E. tenella antigen or 0.i ml of the stock solution of PHA (100 ug) was injected into the right wattle. The left wattle received 0.1 ml PBS. Twenty-four hours after injection, thickness of both the right and left w a t t l e s was measured again and the increase in thickness for each wattle was calculated by subtracting the
Vol. i0, No. 3
WATTLE REACTIONS IN CHICKENS
389
initial thickness from the thickness at twenty-four hours. The twenty-four hour measurement has been shown to be the time of maximum response to antigens (12,13). A~%12-~: Data for each test substance, E. tenella antigen and PHA, were pooled across trials and analyzed by analysis of variance. Group means were compared to the uninfected control using Dunnett's test (21) at the 0.05 level of significance.
RF~ULT~ The D W R to the E. tenella antigen is shown in Figure i. Uninfected chickens showed little reactivity to the antigen demonstrating the specificity of the reaction. On days 4 through i0 post infection, the increase in right wattle thickness of the experimental birds was significantly greater than for the uninfected controls with the highest response being on day 8. The response waned after day 10. The left wattle, injected with saline (data not shown), did not demonstrate a thickness increase significantly different from that of the uninfected controls on any day. [.Or
RT. INCREASE (mm.)
.8
.6
I I .4
.2
0.0 0
t
2
3
4
5 6 7 8 DAYS POST INFECTION FIG 1.
9
I0
11
12
13
Increase in right wattle thickness (ram) following the injection of 0.i ml E. tenella antigen in E_~ tenella infected chickens. Data are represented as mean + standard error. Significant differences (P <0.05) from the control (day 0) are indicated by stars.
390
WATTLE REACTIONS IN CHICKENS
Vol. i0, No. 3
Figure 2 shows the PHA wattle response during the E. tenella infectio~ Beginning on day 3 post infection, wattle responses of the experimental groups were significantly greater than the control except on day 6. An interesting dichotomy of response was observed since those for days 7 through 13 were substantially higher than for those for days 1 through 6. As with the E. tenella antigen, the saline-injected wattles of the experimental groups did not exhibit a significant increase in thickness over that of the salineinjected wattles of the control group. RT. INCREASE
(ram.)
1.4
|.2
l
i
1.0
.8
.6
0
I
2
3
4
5 6 7 B DAYS POST INFECTION
I0
II
12
13
F~.2 Increase in right wattle thickness (mm) following the injection of i00 ug PHA in E. tenella infected chickens. Data are represented as mean + standard error. Significant differences (P <0.05) from the control (day 0) are indicated by stars. DISCUSSION Coccidia are obligate protozoan parasites, primarily of the intestine. Usually there is a rather extensive asexual reproduction within the cells of the mucosa or submucosa, r e s u l t i n g in illness and s o m e t i m e s death. Eimeria tenella has two asexual generations, the first from day 1 to approximately day 3 and the second over the next 2 to 3 days. Following the asexual development, a sexual phase completes the life cycle with the formation of a resistant cyst that, once infective, will initiate parasitism when ingested by another host.
Vol. i0, No. 3
WATTLE REACTIONS IN CHICKENS
391
Previous studies have demonstrated the utility of the delayed wattle reaction (DWR) in assessing the immune response to coccidia (11-13). Giambrone and coworkers (12) showed a positive DWR to Eimeria necatrix antigen 5 weeks after an initial exposure to Cocci Vac R D, (Sterwin, Inc., Opelika, AL) containing sporulated oocysts from 8 species of avian coccida. The response persisted 29 weeks following the primary e:.l~osure at 4 weeks of age, however, booster vaccinations were administered at 18, 22, and 26 weeks of age. The same investigators (22) using tuberculin, diphtheria toxin, or keyhole limpet hemocyanin, found a positive DWR 4-5 days after injection of a dialyzable leukocyte extract, transfer factor, from previously sensitized birds. A similar transfer of DWR to coccidia antigen was also observed (14). The study reported here is the first examining the daily development of the wattle response immediately after exposure to the parasite. Birds inoculated with a single dose of 10,000 E. tenella oocysts showed a significant increase in wattle thickness compared to the control after 4 days. Sensitization occurred during this period. As the life cycle of the parasite progresses, more antigens are available to stimulate immune cells. In addition, sequential repetitive stimulation by parasite antigens may explain the apparent rapid development of the reaction. Tne large increase in the DWR to the E. tenella antigen at day 8 may have been due to the combined effects of sequential antigen stimulation and a lower parasite burden. Rose and Hesketh (1984) observed a similar effect in the lymphocyte blastogenic response to soluble E. tenella antigen. Six days after infection, when the parasite burden was greatest, the blastogenic response was lower than uninfected controls but significantly increased ii days post infection (19). Despite a greater disparity of response between day 6 and day II, their results are similar to the present in vivo study. Different investigators have interpreted various minimum increases in wattle swelling as indicative of a positive response. The antigens used and the method of calculating the increase in wattle swelling have varied from study to study. The assigned value for a positive response has ranged from 0.3 mm increase, for measuring the DWR to BSA (7) to 1.0 mm for measuring the response to coccidia antigen (13). While the study of reactions to BSA used Freund's complete adjuvant in the sensitization protocol, the responses to coccidia antigen in this and previous studies used infection with coccidia without adjuvant. In this study, those thickness increases which were significantly different from the controls all exceeded 0.3 mm. Since saline injected wattles never demonstrated a thickness increase greater than 0.03 mm, saline wattle reactions (SWR) as described by Cotter and coworkers (9), did not occur. The ten fold increase in the antigen injected wattle over the saline injected wattle suggests that a value lower than 1.0 mm, used by Giambrone and associates (13), should be considered positive. This observation is further substantiated by the percentage of positive responses (50%) found in their study (13). No other work has examined the PHA wattle response in coccidia infected chickens. Three days after infection with one dose of i0,000 E. tenella oocysts, the increase in wattle thickness was significantly higher than the uninfected controls. Significant differences persisted throughout the remainder of the study except on day 6 when there was no difference between the infected and uninfected birds. The response to PHA was higher than the response to E. tenella antigen throughout the study. This is not surprising
392
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since the PHA wattle response is nonspecific while that to E. ten ella is antigen specific. It is not known if the same T cell populations are stimulated by P H A a n d E . tenellaantigem However, because of the nonspecific nature, the increase in the PHA response after day 6 post infection may be due to increased sensitivity of T cells or increased release of lymphokines which stimulate responding T cells. Previous work (19) demonstrated in vitro responses to PHA were suppressed during coccidia infection. Suppression began in vitro 3 days after inoculation and reached its maximum when clinical symptoms were greatest. Suppression of mitogenic responses in concert with an active immune response to parasite antigens has been reported (23,24). Plasma from coccidia infected birds suppressed the mitogenic responses of spleen cells taken from uninfected birds . The suppressive activity was found 6 and Ii but not 13 days after infection (19). While complete suppression of the in vivo response to PHA did not occur, thickness increases on days 1-6 were lower than those of days 7-13. This biphasic response may indicate an inhibitory effect of the parasite during the first six days. Both types of wattle tests used in this investigation were effective in measuring the immune response in coccidia infected birds. Further investigations are warranted to assess the responses to different species of coccidia and different exposure doses. ACKNO~ ~ X ~ T S The authors gratefully acknowledge the support of Hoffman-LaRoche, Inc., Nutley, N.J. We thank Drs. W. M. Collins and F. M. McCorkle for critically reviewing the manuscript. Jackie Packard and Margaret Robinson provided excellent secretarial assistance. Scientific Contribution Number 1384 from the NewHampshireAgricultural Experiment Statiom
REFERENCES i.
KLESIUS, P., JC~NSON, W., and KRAMER, T. Delayed wattle reaction as a measure of cell-mediated immunity in the chicken. ~ Sci. 56, 240, 1977.
2.
JANKOVIC, B.D., ISVANESKI, M., MILOSEVIC, D., and POPESKOVIC, L. Delayed hypersensitivity reactions in bursectcmized chickens. Nature 198, 298, 1963.
.
WARNER, N.L., OVARY, Z., and KANTOR, F . S . Delayed hypersensitivity reactions in normal and bursectcmized chickens. I n t . Arch. ~ 40, 719, 1971.
4.
FIEDLER, H., SCHERV, M., KUHLMANN-RUBENS, I., and LOSCH, U. Kinetics of delayed-type hypersensitivity (DHT) reactions and Ig levels in FCAinjected dysgammaglobulinemic BI2 chickens. I _ ~ 158, 293, 19El.
5.
KLIGENSMITH, P.M., DONAHOE, J.P., and STEVENS, J.F. ~he effect of the sex-linked dwarfing gene, dw, on the i~nune responses of broiler breeder chickens. ~ Sci. 62, 733, 1983.
Vol. i0, No. 3
.
7.
.
.
WATTLE REACTIONS IN CHICKENS
393
COffER, M.D., PETERSON, R.D.A., SOUTH, M.A., and GOOD, R.A. The functions of the thymus system and bursa system in the chicken. J. Exp. Med. 123, 75, 1966. PALLADINO, M.A., GREBh-NAU, M.D., and THORBECKE, G.J. Requirements for induction of delayed hypersensitivity in the chicken. Dev. Conp. 2, 121, 1978. WATABE, M., and GLICK, B. The influence of age on the induction of delayed hypersensitivity to human gamma globulin. P _ ~ Sci. 62, 563, 1983. C~, P.F., WING, T., and SWANSON, J. The delayed and saline wattle reactions in broilers challenged with bovine serum albumin. ~ Sci. 64, 1293, 1985.
i0.
BYERLY, Marek's 1972.
J.L., and DAWE, D.L. Delayed hypersensitivity reactions in disease virus-infected chickens. Am. J. Vet. Res. 33, 2267,
ii.
ROSE, M.E. Eimeria tenella: Skin hypersensitivity in the fowl. ExP. ~ 42, 129, 1977.
12.
GIAMBRONE, J.J., KLF~IUS, P.H., and EDGAR, S.A. Avian coccidiosis: Evidence for a cell mediated response. Poultry Sci. 59, 38, 1980.
13.
GIAMBRONE, J.J., AND ~w-SIUS, P . H . Chicken coccidiosis: correlation between resistance and delayed hypersensitivity. ~ Sci. 59, 1715, 1980.
14.
KLESIUS, P.H., and GIA~BRONE, J.J. Adoptive transfer of delayed hypersensitivity and protective inmunity to E ~ r i a tenella with chicken-derived transfer factor. P _ ~ Sci. 63, 1333, 1984.
15.
GOTO, N., KODAMA, H., OKADA, K., and FUJIMOTO, Y. Suppression of phytohemagglutinin skin response in thymectomized chickens. ~ Sci. 57, 246, 1977.
16.
M~RKLE, F.M., STINSON, R., and GLICK, B. A biphasic graft vs. host response in aging chickens. Cell. ~ 46, 209, 1979.
17.
McOORKLE, F., CLAH, I., and GLICK, B. The morphology of the phytohemagglutin-induced cell response in the chicken's wattle. P _ ~ Sci. 59, 616, 1980
18.
OLAH, I., McCORKLE, F., and GLICK, formation in the chicken wattle. P _ ~
19.
ROSE, M.E., and HESKE~H, P. Infection with ~ tenella: Modulation of lymphocyte blastogenesis by specific antigen and evidence for inlnunodepression. J. ~ 31, 549, 1984.
20.
GIAMBRONE, J.J., JOHNSON, L.W., and KLESIUS, P.H. Development of cellmediated i~munity and resistance to clinical coccidiosis infection in chickens selected for resistance and susceptibility to E ~ r i a tenella. P_~ Sci. 63, 2162, 1984.
B.
to injected antigen
Lectin-induced giant Sci. 59, 2151, 1980.
cell
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WATTLE REACTIONS IN CHICKENS
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21.
DUNNE~T, C.W. A multiple comparison procedure for comparing several treatments with a control. J. Am. ~ A s s o c . 50, 1096, 1955.
22.
GIAMBRONE, J.J., KLESIUS, P.H., and YU, M. Adoptive transfer of delayed wattle reactivity in chickens with a dialyzable leukocyte extract containing transfer factor. P_9/ll~Sci. 62, 767, 1983.
23.
VICKERY, A.C., KLEIN, T.W., and FRIEDMAN, H. Modulation of lymphoid cell blastogenic responsiveness to mitogens by NipDostronQvlus infection. Proc. Soc. Exp. Biol. Med. 168, 33, 1981.
24.
WYLER, D.J., and BRf~N, J. Malaria antigen-specific responsiveness during infection with P l a s ~ i ~ ~ C l i n . 29, 401, 1977. Received: December, 1986 Accepted: March, 1986
T-cell ExD.
DELAYED WATILE REACTIONS IN EIMERIA TENk~,TA INFECTED CHICKENS
Robert L. Taylor, Jr., Richard G. Strout, Robert A. Clare and Paul A. Aeed Department of Animal and Nutritional Sciences University of New Hampshire Durham, New Hampshire USA 03824
ABSTRACT
Wattle reactions to a n ~ t e n e l l a antigen and phytohemagglutinin (PHA) were studied in chickens infected with E. tenella. Two trials were conducted using a total of 224 chickens. Four days after infection with one dose of i0,000 sporulated oocysts, the increase in wattle thickness in response to E. tenella antigen was significantly greater than that of uninfected controls. This significant response persisted through day i0 post infectio~ Wattle responses to PHA 3 days after infection were significantly greater than for uninfected controls. Significant differences in response to PHA were maintained throughout the experiment except on day 6. An increased response to PHA from days 7 to 13 post infection was attributed to the lower parasite burden at that time.
INTRODUCTION Cellular immunity in chickens has been assessed by delayed wattle reactions (DWR) (i). In this technique, sensitized birds are injected intradermally in the wattle with the sensitizing antigen and the resulting swelling in the injected wattle is then used to determine the degree of response. Such tests are performed easily and have been correlated with other tests of T cell function such as spleen cell migration inhibition and lymphocyte blastogenic responses (i). Antigens t e s t e d in this s y s t e m i n c l u d e t u b e r c u l i n (2-5), dinitrophenylated chicken serum albumin (3), diphtheria toxin (1,6), human g a m m a globulin (7,8), and bovine serum albumin (7,9). Disease organisms tested include S a l m o n e l l a adelaide (3), Marek's disease virus (i0) and coccidia (11-14). Phytohemagglutinin (PHA), a mitogenic lectin extracted from v~laaris, has also been used in wattle tests. This reaction proceeds without prior sensitization and is mediated by T cells (15,16). The first cells to 387
388
WATTLE REACTIONS IN CHICKENS
Vol. i0, No. 3
infiltrate the wattle were heterophils and mononuclear cells in as little as 30 min. followed by basophils and eosinophils 24 hours after injection. Granulocyte numbers declined by 48 hours while lymphocytes increased (17). Multinucleated giant cells were also observed (18) in the region of infiltration. Rose and Hesketh (1984) reported that infection with Eimeria tenella depressed the T lymphocyte blastogenic response to PHA (19). In contrast, the T cell response to E. tenella antigen increased during the course of parasitism. Giambrone and coworkers (20) found a high correlation between innate resistance and DWR in lines selected for susceptibility and resistance to coccidiosis. Their tests were performed 2 to 6 weeks after initial exposure. The DWR to coccidia antigens during the time immediately following infection has not been investigated. This paper reports the wattle responses to an antigen from E. tenella and to PHA in birds during the first 13 days after infection with E. tenella.
MATERIAL S AhD M E ~ O D S Animals: Straight run Single Comb White Leghorns were used throughout this study. Birds were maintained according to standard management practices at the University of New Hampshire Poultry Research Farn~ The chicks were raised coccidia-free in heated metal batteries with free access to unmedicated feed and water. Vaccinations against Marek's disease and Newcastle-bronchitis were administered during the growing period. Coccidia cultures: The Lilly 65 strain of Eimeria tenella (Eli Lilly, Greenfield, IN9 was maintained in our laboratory. Viability and pathogenicity were preserved by serial passage at regular intervals. Suspensions of counted oocysts were administered to each chicken orally using an inoculation tube and syringe. ~Jl~q~: Sterile oocysts were suspended in 20 m~ sterile phosphate buffered saline (PBS, pH 7.2) at a concentration of 10 v oocysts/ml. The oocyst suspension was twice disrupted at 10,000 PSI in a sterile French Press (Fred S. Carver, Inc., Summit, NJ) and subjected to two freeze-thaw cycles. The antigen preparation was kept frozen until use. lli~: A stock solution containing 1 mg/ml of phytohemagglutinin (PHA-P, Difco Laboratories, Detroit, MI) was prepared in sterile PBS (pH 7.2). Wattle test: TWo trials, each consisting of 112 animals, gave a total of 224 chickens. A t six weeks of age, birds were randomly divided into 14 groups of 8 birds each. Group 1 (Day 0) was designated the uninfected control. The remaining 13 groups received a single inoculum of 10,000 sporulated oocysts of Eimeria ~ On each of 14 consecutive days, birds from one group were tested for their wattle reaction to either E. tenella antigen or PHA. The wattle test was conducted by measuring the thickness of each wattle with a micrometer. After measurement, either 0~I ml of the E. tenella antigen or 0.i ml of the stock solution of PHA (100 ug) was injected into the right wattle. The left wattle received 0.1 ml PBS. Twenty-four hours after injection, thickness of both the right and left w a t t l e s was measured again and the increase in thickness for each wattle was calculated by subtracting the
Vol. i0, No. 3
WATTLE REACTIONS IN CHICKENS
389
initial thickness from the thickness at twenty-four hours. The twenty-four hour measurement has been shown to be the time of maximum response to antigens (12,13). A~%12-~: Data for each test substance, E. tenella antigen and PHA, were pooled across trials and analyzed by analysis of variance. Group means were compared to the uninfected control using Dunnett's test (21) at the 0.05 level of significance.
RF~ULT~ The D W R to the E. tenella antigen is shown in Figure i. Uninfected chickens showed little reactivity to the antigen demonstrating the specificity of the reaction. On days 4 through i0 post infection, the increase in right wattle thickness of the experimental birds was significantly greater than for the uninfected controls with the highest response being on day 8. The response waned after day 10. The left wattle, injected with saline (data not shown), did not demonstrate a thickness increase significantly different from that of the uninfected controls on any day. [.Or
RT. INCREASE (mm.)
.8
.6
I I .4
.2
0.0 0
t
2
3
4
5 6 7 8 DAYS POST INFECTION FIG 1.
9
I0
11
12
13
Increase in right wattle thickness (ram) following the injection of 0.i ml E. tenella antigen in E_~ tenella infected chickens. Data are represented as mean + standard error. Significant differences (P <0.05) from the control (day 0) are indicated by stars.
390
WATTLE REACTIONS IN CHICKENS
Vol. i0, No. 3
Figure 2 shows the PHA wattle response during the E. tenella infectio~ Beginning on day 3 post infection, wattle responses of the experimental groups were significantly greater than the control except on day 6. An interesting dichotomy of response was observed since those for days 7 through 13 were substantially higher than for those for days 1 through 6. As with the E. tenella antigen, the saline-injected wattles of the experimental groups did not exhibit a significant increase in thickness over that of the salineinjected wattles of the control group. RT. INCREASE
(ram.)
1.4
|.2
l
i
1.0
.8
.6
0
I
2
3
4
5 6 7 B DAYS POST INFECTION
I0
II
12
13
F~.2 Increase in right wattle thickness (mm) following the injection of i00 ug PHA in E. tenella infected chickens. Data are represented as mean + standard error. Significant differences (P <0.05) from the control (day 0) are indicated by stars. DISCUSSION Coccidia are obligate protozoan parasites, primarily of the intestine. Usually there is a rather extensive asexual reproduction within the cells of the mucosa or submucosa, r e s u l t i n g in illness and s o m e t i m e s death. Eimeria tenella has two asexual generations, the first from day 1 to approximately day 3 and the second over the next 2 to 3 days. Following the asexual development, a sexual phase completes the life cycle with the formation of a resistant cyst that, once infective, will initiate parasitism when ingested by another host.
Vol. i0, No. 3
WATTLE REACTIONS IN CHICKENS
391
Previous studies have demonstrated the utility of the delayed wattle reaction (DWR) in assessing the immune response to coccidia (11-13). Giambrone and coworkers (12) showed a positive DWR to Eimeria necatrix antigen 5 weeks after an initial exposure to Cocci Vac R D, (Sterwin, Inc., Opelika, AL) containing sporulated oocysts from 8 species of avian coccida. The response persisted 29 weeks following the primary e:.l~osure at 4 weeks of age, however, booster vaccinations were administered at 18, 22, and 26 weeks of age. The same investigators (22) using tuberculin, diphtheria toxin, or keyhole limpet hemocyanin, found a positive DWR 4-5 days after injection of a dialyzable leukocyte extract, transfer factor, from previously sensitized birds. A similar transfer of DWR to coccidia antigen was also observed (14). The study reported here is the first examining the daily development of the wattle response immediately after exposure to the parasite. Birds inoculated with a single dose of 10,000 E. tenella oocysts showed a significant increase in wattle thickness compared to the control after 4 days. Sensitization occurred during this period. As the life cycle of the parasite progresses, more antigens are available to stimulate immune cells. In addition, sequential repetitive stimulation by parasite antigens may explain the apparent rapid development of the reaction. Tne large increase in the DWR to the E. tenella antigen at day 8 may have been due to the combined effects of sequential antigen stimulation and a lower parasite burden. Rose and Hesketh (1984) observed a similar effect in the lymphocyte blastogenic response to soluble E. tenella antigen. Six days after infection, when the parasite burden was greatest, the blastogenic response was lower than uninfected controls but significantly increased ii days post infection (19). Despite a greater disparity of response between day 6 and day II, their results are similar to the present in vivo study. Different investigators have interpreted various minimum increases in wattle swelling as indicative of a positive response. The antigens used and the method of calculating the increase in wattle swelling have varied from study to study. The assigned value for a positive response has ranged from 0.3 mm increase, for measuring the DWR to BSA (7) to 1.0 mm for measuring the response to coccidia antigen (13). While the study of reactions to BSA used Freund's complete adjuvant in the sensitization protocol, the responses to coccidia antigen in this and previous studies used infection with coccidia without adjuvant. In this study, those thickness increases which were significantly different from the controls all exceeded 0.3 mm. Since saline injected wattles never demonstrated a thickness increase greater than 0.03 mm, saline wattle reactions (SWR) as described by Cotter and coworkers (9), did not occur. The ten fold increase in the antigen injected wattle over the saline injected wattle suggests that a value lower than 1.0 mm, used by Giambrone and associates (13), should be considered positive. This observation is further substantiated by the percentage of positive responses (50%) found in their study (13). No other work has examined the PHA wattle response in coccidia infected chickens. Three days after infection with one dose of i0,000 E. tenella oocysts, the increase in wattle thickness was significantly higher than the uninfected controls. Significant differences persisted throughout the remainder of the study except on day 6 when there was no difference between the infected and uninfected birds. The response to PHA was higher than the response to E. tenella antigen throughout the study. This is not surprising
392
WATTLE REACTIONS IN CHICKENS
Vol. i0, No. 3
since the PHA wattle response is nonspecific while that to E. ten ella is antigen specific. It is not known if the same T cell populations are stimulated by P H A a n d E . tenellaantigem However, because of the nonspecific nature, the increase in the PHA response after day 6 post infection may be due to increased sensitivity of T cells or increased release of lymphokines which stimulate responding T cells. Previous work (19) demonstrated in vitro responses to PHA were suppressed during coccidia infection. Suppression began in vitro 3 days after inoculation and reached its maximum when clinical symptoms were greatest. Suppression of mitogenic responses in concert with an active immune response to parasite antigens has been reported (23,24). Plasma from coccidia infected birds suppressed the mitogenic responses of spleen cells taken from uninfected birds . The suppressive activity was found 6 and Ii but not 13 days after infection (19). While complete suppression of the in vivo response to PHA did not occur, thickness increases on days 1-6 were lower than those of days 7-13. This biphasic response may indicate an inhibitory effect of the parasite during the first six days. Both types of wattle tests used in this investigation were effective in measuring the immune response in coccidia infected birds. Further investigations are warranted to assess the responses to different species of coccidia and different exposure doses. ACKNO~ ~ X ~ T S The authors gratefully acknowledge the support of Hoffman-LaRoche, Inc., Nutley, N.J. We thank Drs. W. M. Collins and F. M. McCorkle for critically reviewing the manuscript. Jackie Packard and Margaret Robinson provided excellent secretarial assistance. Scientific Contribution Number 1384 from the NewHampshireAgricultural Experiment Statiom
REFERENCES i.
KLESIUS, P., JC~NSON, W., and KRAMER, T. Delayed wattle reaction as a measure of cell-mediated immunity in the chicken. ~ Sci. 56, 240, 1977.
2.
JANKOVIC, B.D., ISVANESKI, M., MILOSEVIC, D., and POPESKOVIC, L. Delayed hypersensitivity reactions in bursectcmized chickens. Nature 198, 298, 1963.
.
WARNER, N.L., OVARY, Z., and KANTOR, F . S . Delayed hypersensitivity reactions in normal and bursectcmized chickens. I n t . Arch. ~ 40, 719, 1971.
4.
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T-cell ExD.