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The Delayed and Saline Wattle Reactions in Broilers Challenged with Bovine Serum Albumin
The Delayed and Saline Wattle Reactions in Broilers Challenged with Bovine Serum Albumin p. F. COTTER1 Department of Biology, Framingham State College, Framingham, Massachusetts 01701 T. WING and J. SWANSON Cobb Incorporated, Concord, Massachusetts 01742 (Received for publication July 19, 1984) ABSTRACT Bovine serum albumin was used to study the development of a wattle reaction in broiler-type chickens. A strong response developed in both nonsensitized and sensitized chickens and the frequency of such was shown to be dependent on the method used for the sensitization. A similar swelling developed in a proportion of the wattles injected with saline and this too seemed to be influenced by the sensitization method. Moreover, both types of responses were shown to be somewhat unstable during the first 24 hr postchallenge. (Key words: delayed wattle reaction, saline wattle reaction) 1985 Poultry Science 64:1293-1295
INTRODUCTION T h e delayed wattle reaction (DWR) is often used as an indication of a cell-mediated i m m u n e response in chickens (Klesius et al.,1977). It is usually measured b y noting thickness increases in t h e test wattles of sensitized chickens after a challenge injection with the sensitizing imm u n o g e n . S o m e investigators have described a DWR t h a t occurs in nonsenstized chickens challenged directly w i t h phytohemagglutinin ( G o t o et al, 1 9 7 8 ; Mashaly, 1 9 8 4 ) . Others, Palladino et al. ( 1 9 7 8 ) , have used b o t h intramuscular or intravenous sensitization t o study t h e developm e n t of t h e DWR challenge injections w i t h bovine serum albumin. However, little a t t e n t i o n has been given t o the effects t h a t t h e sensitization p r o t o c o l has on the frequency of positive DWR in broilers or t o a DWR-like response t h a t sometimes occurs in t h e saline-injected control wattle. We have called this latter response t h e "saline w a t t l e r e a c t i o n " (SWR) (Cotter et al, 1 9 8 4 ) . Here w e r e p o r t d a t a t h a t shows h o w t h e sensitization p r o t o c o l can effect b o t h the DWR and SWR. MATERIALS AND METHODS This s t u d y was conducted using male com-
1
Dedicated to Kathleen and to the memory of Mary M. Cotter.
mercial broiler-type chickens (Cobb) 8 t o 10 weeks of age. T h e y were sensitized w i t h bovine serum albumin (BSA) (Cohn fraction V, Sigma) by intramuscular breast injection or b y foot pad injection. Each animal received 2 0 0 0 jJLg as a result of either m e t h o d . Other chickens of t h e same group were n o t sensitized. All animals were challenged, 6 days later, in one w a t t l e with 1 0 0 0 ^g BSA given in a t o t a l v o l u m e of .1 ml. T h e other wattle received t h e same a m o u n t of saline. Measurements of w a t t l e thickness were t a k e n prior t o the challenge and at 1 a n d 2 4 hr postchallenge. A positive response required a thickness increase in t h e test wattle of at least .6 m m w h e n c o m p a r e d w i t h the prechallenge value. T h e SWR was measured similarly. Statistical differences b e t w e e n sensitized a n d nonsensitized groups w e r e d e t e r m i n e d by chi square (Zar, 1 9 8 4 ) . RESULTS T h e frequency of positive DWR t o bovine serum albumin is d e p e n d e n t u p o n t h e m e t h o d used for t h e sensitization (Table 1). Significant differences were detected b e t w e e n all t r e a t m e n t groups ( x 2 = 2 7 . 3 , P < . 0 0 1 ) , which suggests t h a t sensitization m e t h o d s , in general, result in a higher incidence of positive DWR. F u r t h e r m o r e , t h e incidence of positive reactions at 1 hr in t h e foot pad-sensitized g r o u p was significantly different from t h e 24-hr incidence, indi-
1293
1294
COTTER ET AL. TABLE 1. The delayed wattle reaction in broilers challenged with bovine serum albumin Hours postchallenge
method
1
None Intramuscular Foot pad
5/232,i 7/15b 18/20 c
24 5/222 7/15 b 12/20 b
22% 48% 90%
24% 48% 60%
' ' Groups with the same superscript are statistically equivalent (P<.05). 1
The numerator is the number of positive responses; the denominator is the number tested.
eating that an interaction may occur during development of the DWR. These same data also show that sensitization is not an absolute prerequisite for the development of a positive response to BSA, because approximately 20% of the nonsensitized group also developed such reactions. A reaction mimicking the DWR can occur in the saline-injected wattle (Table 2). The SWR occurred in at least some members of each group and it also seemed dependent upon sensitization method (x 2 = 28.1, P<.001). The SWR may develop immediately after the challenge or may appear later, as is shown in the group sensitized by the foot pad technique. Like the DWR, the presence of an SWR is not absolutely dependent upon prior sensitization, because it occurred in the nonsensitized group at both 1 and 24 hr after the challenge. The data presented in Table 3 show that the DWR response is not necessarily stable over the first 24 hr. Although the majority of such reactions (63%) do not change over this period, changes from positive to negative (26%) and negative to positive (11%) can occur within this time.
DISCUSSION
Our data indicate that broiler-type chickens may develop a strong wattle response to a challenge injection with bovine serum albumin. Furthermore, the development of the response is often immediate, reaching a significant (.6 mm) value within 1 hr postchallenge (Klesius et al., 1977). Certain workers have attributed this early response to an immediate type hypersensitivity and have chosen to remove such animals from further study of the DWR (Palladino et al, 1978; Klingensmith etal, 1983). In all likelihood the wattle reactions described here are the result of a mixed response, the nature of which could be influenced by such variables as the type of the immunogen used, its route of presentation, and genetic differences present in the chickens studied. Indeed, this latter point has been established by Krejci et al. (1974), who have shown differences in the development of tuberculin hypersensitivity between inbred lines. The SWR may also be a manifestation of such a mixed response. Perhaps some physiologically active, low molecular weight substances, released by the reacting cells in the test
TABLE 2. The salimi wattle reaction in broilers challenged with bovine serum alb,umin Hours postchallenge metiiod None Intramuscular Foot pad
24
1 3/232,i
6/15 b 3/202
13% 40% 15%
1/222 2/152 13/20 c
' ' Groups with the same superscript are statistically equivalent (P<.05). 1
The numerator is the number of positive responses; the denominator is the number tested.
5% 13% 65%
DELAYED AND SALINE WATTLE REACTIONS IN BROILERS
1295
TABLE 3. The change in the delayed wattle reaction between 1 and 24 hr postchullenge,• (0) = no change, (—/+) = negative at 1 hr, positive at 24 hr; (+/—) = positive at 1 hr, negative at 24 hr Sensitization method
(0)
None Intramuscular Footpad
15 1 12 9
Totals
36/57 63%
1
(-/+) 3 1 2 6/57 11%
(+/-) 4 2 9 15/57 26%
One animal had a resolution of the test wattle while die saline wattle remained positive.
wattle, can diffuse into the saline-injected wattle where they cause the observed swellings. The resolution of this reaction may then depend upon the later arrival of immunoregulatory cells, presumably derived from the thymus (Cooper et al., 1966). Alternatively, local tissue conditions present in the saline-injected wattle may cause a gradual inactivation of the diffusing substance(s) in the absence of specifically committed immunocompetent cells. Both innate and acquired resistance to avian coccidiosis may depend upon the cell-mediated immune response, and recently the DWR has been used to predict such resistance in Leghorns (Giambrone et al, 1984). Thus, additional information on the development and regulation of the DWR and the related SWR may provide further insight into the generalized nature of both types of resistance. Moreover, genetic information regarding the nature of either response may be of use in development of improved disease resistance through selective breeding. ACKNOWLEDGMENT The authors wish to thank Tom Haight for help and advice with the statistical analysis.
REFERENCES Cooper, M. D., R.D.A. Peterson, M. A. South, and
R. A. Good, 1966. The functions of the thymus system and the bursa system in the chicken. J. Exp. Med. 123:75-102. Cotter, P. F., T. Wing, and J. Swanson, 1984. The development and measurement of the delayed wattle reaction in broilers. Poultry Sci. 63:82. (Abstr.) Giambrone, J. J., L. W. Johnson, and P. H. Klesius, 1984. Development of cell-mediated immunity and resistance to clinical coccidiosis infection in chickens selected for resistance and susceptibility to Eimeria tenella. Poultry Sci. 63:2162— 2166. Goto, N., H. Kodama, K. Okada, and Y. Fujimoto, 1978. Suppression of phytohemagglutinin skin response in thymectomized chickens. Poultry Sci. 57:246-250. Klesius, P., W, Johnson, and T. Kramer, 1977. Delayed wattle reaction as a measure of cell-mediated immunity in the chicken. Poultry Sci. 56:249—256. Klingensmith, P. M., J. P. Donahoe, and J. F. Stevens, 1983. The effect of the sex-linked dwarfing gene, dw, on the immune responses of broiler breeder chickens. Poultry Sci. 62:733-740. Krejci, J., I. Karakoz, J. Pekarek, T. Hraba, and K. Hula, 1974. Differences between inbred lines of chickens in development of tuberculin hypersensitivity. Immunology 27:133—136. Mashaly, M. M., 1984. Effect of caponization on cellmediated immunity of immature cockerels. Poultry Sci. 63:369-372. Palladino, M. M., M. D. Grebenau, and G. J. Thorbecke, 1978. Requirements for induction of delayed hypersensitivity in the chicken. Dev. Comp. Immunol. 2:121-132. Zar, J. H., 1984. Pages 4 0 0 - 4 0 2 in Biostatistical Analysis. 2nd ed. Prentice-Hall, Inc., Englewood Cliffs, NJ.
INTRODUCTION T h e delayed wattle reaction (DWR) is often used as an indication of a cell-mediated i m m u n e response in chickens (Klesius et al.,1977). It is usually measured b y noting thickness increases in t h e test wattles of sensitized chickens after a challenge injection with the sensitizing imm u n o g e n . S o m e investigators have described a DWR t h a t occurs in nonsenstized chickens challenged directly w i t h phytohemagglutinin ( G o t o et al, 1 9 7 8 ; Mashaly, 1 9 8 4 ) . Others, Palladino et al. ( 1 9 7 8 ) , have used b o t h intramuscular or intravenous sensitization t o study t h e developm e n t of t h e DWR challenge injections w i t h bovine serum albumin. However, little a t t e n t i o n has been given t o the effects t h a t t h e sensitization p r o t o c o l has on the frequency of positive DWR in broilers or t o a DWR-like response t h a t sometimes occurs in t h e saline-injected control wattle. We have called this latter response t h e "saline w a t t l e r e a c t i o n " (SWR) (Cotter et al, 1 9 8 4 ) . Here w e r e p o r t d a t a t h a t shows h o w t h e sensitization p r o t o c o l can effect b o t h the DWR and SWR. MATERIALS AND METHODS This s t u d y was conducted using male com-
1
Dedicated to Kathleen and to the memory of Mary M. Cotter.
mercial broiler-type chickens (Cobb) 8 t o 10 weeks of age. T h e y were sensitized w i t h bovine serum albumin (BSA) (Cohn fraction V, Sigma) by intramuscular breast injection or b y foot pad injection. Each animal received 2 0 0 0 jJLg as a result of either m e t h o d . Other chickens of t h e same group were n o t sensitized. All animals were challenged, 6 days later, in one w a t t l e with 1 0 0 0 ^g BSA given in a t o t a l v o l u m e of .1 ml. T h e other wattle received t h e same a m o u n t of saline. Measurements of w a t t l e thickness were t a k e n prior t o the challenge and at 1 a n d 2 4 hr postchallenge. A positive response required a thickness increase in t h e test wattle of at least .6 m m w h e n c o m p a r e d w i t h the prechallenge value. T h e SWR was measured similarly. Statistical differences b e t w e e n sensitized a n d nonsensitized groups w e r e d e t e r m i n e d by chi square (Zar, 1 9 8 4 ) . RESULTS T h e frequency of positive DWR t o bovine serum albumin is d e p e n d e n t u p o n t h e m e t h o d used for t h e sensitization (Table 1). Significant differences were detected b e t w e e n all t r e a t m e n t groups ( x 2 = 2 7 . 3 , P < . 0 0 1 ) , which suggests t h a t sensitization m e t h o d s , in general, result in a higher incidence of positive DWR. F u r t h e r m o r e , t h e incidence of positive reactions at 1 hr in t h e foot pad-sensitized g r o u p was significantly different from t h e 24-hr incidence, indi-
1293
1294
COTTER ET AL. TABLE 1. The delayed wattle reaction in broilers challenged with bovine serum albumin Hours postchallenge
method
1
None Intramuscular Foot pad
5/232,i 7/15b 18/20 c
24 5/222 7/15 b 12/20 b
22% 48% 90%
24% 48% 60%
' ' Groups with the same superscript are statistically equivalent (P<.05). 1
The numerator is the number of positive responses; the denominator is the number tested.
eating that an interaction may occur during development of the DWR. These same data also show that sensitization is not an absolute prerequisite for the development of a positive response to BSA, because approximately 20% of the nonsensitized group also developed such reactions. A reaction mimicking the DWR can occur in the saline-injected wattle (Table 2). The SWR occurred in at least some members of each group and it also seemed dependent upon sensitization method (x 2 = 28.1, P<.001). The SWR may develop immediately after the challenge or may appear later, as is shown in the group sensitized by the foot pad technique. Like the DWR, the presence of an SWR is not absolutely dependent upon prior sensitization, because it occurred in the nonsensitized group at both 1 and 24 hr after the challenge. The data presented in Table 3 show that the DWR response is not necessarily stable over the first 24 hr. Although the majority of such reactions (63%) do not change over this period, changes from positive to negative (26%) and negative to positive (11%) can occur within this time.
DISCUSSION
Our data indicate that broiler-type chickens may develop a strong wattle response to a challenge injection with bovine serum albumin. Furthermore, the development of the response is often immediate, reaching a significant (.6 mm) value within 1 hr postchallenge (Klesius et al., 1977). Certain workers have attributed this early response to an immediate type hypersensitivity and have chosen to remove such animals from further study of the DWR (Palladino et al, 1978; Klingensmith etal, 1983). In all likelihood the wattle reactions described here are the result of a mixed response, the nature of which could be influenced by such variables as the type of the immunogen used, its route of presentation, and genetic differences present in the chickens studied. Indeed, this latter point has been established by Krejci et al. (1974), who have shown differences in the development of tuberculin hypersensitivity between inbred lines. The SWR may also be a manifestation of such a mixed response. Perhaps some physiologically active, low molecular weight substances, released by the reacting cells in the test
TABLE 2. The salimi wattle reaction in broilers challenged with bovine serum alb,umin Hours postchallenge metiiod None Intramuscular Foot pad
24
1 3/232,i
6/15 b 3/202
13% 40% 15%
1/222 2/152 13/20 c
' ' Groups with the same superscript are statistically equivalent (P<.05). 1
The numerator is the number of positive responses; the denominator is the number tested.
5% 13% 65%
DELAYED AND SALINE WATTLE REACTIONS IN BROILERS
1295
TABLE 3. The change in the delayed wattle reaction between 1 and 24 hr postchullenge,• (0) = no change, (—/+) = negative at 1 hr, positive at 24 hr; (+/—) = positive at 1 hr, negative at 24 hr Sensitization method
(0)
None Intramuscular Footpad
15 1 12 9
Totals
36/57 63%
1
(-/+) 3 1 2 6/57 11%
(+/-) 4 2 9 15/57 26%
One animal had a resolution of the test wattle while die saline wattle remained positive.
wattle, can diffuse into the saline-injected wattle where they cause the observed swellings. The resolution of this reaction may then depend upon the later arrival of immunoregulatory cells, presumably derived from the thymus (Cooper et al., 1966). Alternatively, local tissue conditions present in the saline-injected wattle may cause a gradual inactivation of the diffusing substance(s) in the absence of specifically committed immunocompetent cells. Both innate and acquired resistance to avian coccidiosis may depend upon the cell-mediated immune response, and recently the DWR has been used to predict such resistance in Leghorns (Giambrone et al, 1984). Thus, additional information on the development and regulation of the DWR and the related SWR may provide further insight into the generalized nature of both types of resistance. Moreover, genetic information regarding the nature of either response may be of use in development of improved disease resistance through selective breeding. ACKNOWLEDGMENT The authors wish to thank Tom Haight for help and advice with the statistical analysis.
REFERENCES Cooper, M. D., R.D.A. Peterson, M. A. South, and
R. A. Good, 1966. The functions of the thymus system and the bursa system in the chicken. J. Exp. Med. 123:75-102. Cotter, P. F., T. Wing, and J. Swanson, 1984. The development and measurement of the delayed wattle reaction in broilers. Poultry Sci. 63:82. (Abstr.) Giambrone, J. J., L. W. Johnson, and P. H. Klesius, 1984. Development of cell-mediated immunity and resistance to clinical coccidiosis infection in chickens selected for resistance and susceptibility to Eimeria tenella. Poultry Sci. 63:2162— 2166. Goto, N., H. Kodama, K. Okada, and Y. Fujimoto, 1978. Suppression of phytohemagglutinin skin response in thymectomized chickens. Poultry Sci. 57:246-250. Klesius, P., W, Johnson, and T. Kramer, 1977. Delayed wattle reaction as a measure of cell-mediated immunity in the chicken. Poultry Sci. 56:249—256. Klingensmith, P. M., J. P. Donahoe, and J. F. Stevens, 1983. The effect of the sex-linked dwarfing gene, dw, on the immune responses of broiler breeder chickens. Poultry Sci. 62:733-740. Krejci, J., I. Karakoz, J. Pekarek, T. Hraba, and K. Hula, 1974. Differences between inbred lines of chickens in development of tuberculin hypersensitivity. Immunology 27:133—136. Mashaly, M. M., 1984. Effect of caponization on cellmediated immunity of immature cockerels. Poultry Sci. 63:369-372. Palladino, M. M., M. D. Grebenau, and G. J. Thorbecke, 1978. Requirements for induction of delayed hypersensitivity in the chicken. Dev. Comp. Immunol. 2:121-132. Zar, J. H., 1984. Pages 4 0 0 - 4 0 2 in Biostatistical Analysis. 2nd ed. Prentice-Hall, Inc., Englewood Cliffs, NJ.