Effect of Reserpine on Humoral Immune Responsiveness in Young Chickens12'3 F . W . E D E N S , C . V . S I K E S , J R . , P . THAXTON AND G . W . MORGAN, J R .
Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27607 (Received for publication March 11, 1975)
POULTRY SCIENCE 54: 1970-1975, 1975
INTRODUCTION
R
ESERPINE, which is used widely as an anti-hypertensive and tranquilizing agent, has been used as an anti-stress compound in chickens (see Edens and Siegel, 1974a). Reserpine acts at the level of the adrenal medulla and at post-ganglionic sympathetic nerve endings where the stores of the catecholamines are depleted by inhibition of uptake of dopamine and norepinephrine (Koelle, 1970). Additionally, reserpine is reported to cause a rise in the circulating levels of corticosterone in both mammals (Montanari and Stockham, 1962) and chickens (Srebocan et al., 1972; Edens and Siegel, 1974b). These reports suggest that this rise following reserpine treatment is the result of a concerted mobilization of hypophysial ACTH reserves. Exogenous ACTH is thought to suppress humoral immune responsiveness by stimulating the secretion of the adreno-
1. Paper Number 4606 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina. 2. A preliminary report of this paper was presented to the 63rd Annual Meeting of the Poultry Science Association, Morgantown, West Virginia. 3. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Experiment Station of the product named nor criticism of similar ones not mentioned.
cortical steriods (Thaxton et al., 1968; Subba Rao and Glick, 1970; Thaxton, 1971; Thaxton and Siegel, 1973). Melmon etal. (1974a, b) and Bourne et al. (1974) demonstrated that humoral antibody production is also suppressed by the catecholamines. Although both the catecholamines and adrenocortical steriods are reported to suppress humoral immune responses, their influence on immunity when considered collectively has not been reported. Thus, the objective of the present study was to determine the effects of reserpine on humoral immune responsiveness and to ascertain how the total adrenal may influence immune responsiveness in chickens. MATERIALS AND METHODS Cobb x Arbor Acre cockerels were used in three trials. The chicks were reared in heated brooding batteries until three weeks of age. Thereafter they were maintained in non-heated metal cages. A broiler startergrower ration and water were available ad libitum. In Trials 1 and 2 the chickens were randomized into seven treatment groups consisting of 10 chickens per group. When the chicks were four weeks of age reserpine, 4 4. Reserpine (3, 4, 5-trimethoxy-benzoyl methyl reserpate) obtained from Sigma Chemical Company, St. Louis, MO.
1970
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on June 11, 2015
ABSTRACT The role of reserpine in modifying a primary humoral immune response was evaluated in three experiments with young chickens. It was found that chickens injected with reserpine prior to an intravenous antigenic challenge with sheep red blood cells exhibited an enhanced primary humoral immune response. The enhanced response occurred concomitant with an elevation in adrenal cortical activity as evidenced by the significant elevation of serum corticosterone in the reserpine-treated chickens. These data suggest that both the adrenal cortex and medulla may influence immune responsiveness in the chicken.
1971
RESERPINE AND HUMORAL IMMUNITY
analysis of corticosterone at the following times: 1) immediately prior to the antigen challenge, 2) at 12 hour intervals for the first two days following the antigen and 3) daily thereafter for the next five days. Serum samples in all of the trials were collected according to the procedure described by Thaxton and Siegel (1973). The samples were assayed serologically by a microtitration procedure (Witlin, 1967) to determine the anti-SRBC antibody levels. Each sample was assayed by the competitive protein binding procedure of Murphy (1967) to determine the level of corticosterone. Nested analyses of variance were performed on all data according to Ostle (1963) and treatment differences were assigned by Kramer's (1956) modification of Duncan's multiple range test. Statements of significance are based on differences at the 5% level of probability. In Trials 1 and 2 no replicate differences were observed, therefore the data from these trials were pooled and are presented collectively. Trial 3 was of a different design and therefore it was analyzed and presented separately.
RESULTS Antibody Responses. The data illustrating the effects of reserpine on humoral immune responsiveness in the birds of Trials 1 and 2 are presented in Table 1. The mean antiSRBC antibody levels in birds receiving reserpine, regardless of time of administration, were higher than those of the gelatin or saline groups during the first six days of the primary hemagglutinin response. Antibody levels of the reserpine-treated birds were significantly higher than those of the gelatin and saline groups on both day four and day six. There were no significant differences among the treatment groups on day eight. These data indicate that the magnitude of the primary response was enhanced by the action of reserpine, but this enhancement appeared to
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on June 11, 2015
which was suspended in a 4% gelatin vehicle, was injected in a single intramuscular dose at the level of 0.75 mg./kg. of body weight. Three groups of birds were given reserpine at 12,6, or 0 hours prior to a single intravenous challenge with 1 ml. of a 10% saline suspension of sheep red blood cells (SRBC). Another three groups were designated as gelatin vehicle controls. They were injected intramuscularly with the 4% gelatin vehicle at the dose level of 1 ml. /kg. of body weight. These injections were given on the same schedule as the reserpine treatments. The final group of chickens, which served as saline controls, were given a single intramuscular injection of physiological saline at the level of 1 ml. /kg. of body weight. This injection was given 12 hours prior to the antigen. All the birds in Trials 1 and 2 were bled by venipuncture prior to the antigen challenge and thereafter at two day intervals for the next eight days to serologically monitor the ensuing primary hemagglutinin response. Following the final bleeding all the birds were weighed, killed by cervical dislocation, and the adrenal glands were excised and weighed. The adrenals were frozen (—20° C.) for subsequent analysis of corticosterone (Guillemin et al., 1959). In Trial 3, four week old cockerels were randomized into three groups of 40 birds each. Two groups were given reserpine (0.75 mg. /kg. of body weight) at 6 or 0 hours prior to antigen challenge with SRBC. The third or control group was injected with the gelatin vehicle at 0 hours prior to antigenic challenge. Differences between the saline controls and gelatin vehicle controls of Trials 1 and 2 were not observed, thus the saline controls were not used in Trial 3. To minimize the possibility of a handling stress from serial bleedings four birds, which were selected at random from each of the three groups, were used at each measurement interval. The birds were bled, killed, and the adrenals were collected for later chemical
1972
EDENS, SIKES, JR., THAXTON AND MORGAN, JR.
TABLE 1.—The mean> (±S.E.) anti-sheep red blood cell (SRBC) antibody levels (log2) for Trials 1 and 2
be transitory since there were no treatment differences at day eight. The antibody response data of the birds of Trial 3 are presented graphically in Figure 1. This trial was conducted to determine if reserpine shortened the response time for peak antibody titers. This result was suggested by the data of Trials 1 and 2. The results of Trial 3 indicate that the administration of reserpine prior to anti6 HOUR RESERPINE OHOUR RESERPINE OHOUR GELATIN
8
— —
hi > hi
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6-
u
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a
r
i
0 1 2 3 4 S 6 7 DAYS POST CHALLENGE WITH SRBC FIG. 1. Anti-SRBC antibody responses in chickens given either reserpine at 6 or 0 hours prior to the SRBC antigen challenge or the gelatin vehicle at 0 hours prior to the SRBC antigen challenge.
genie challenge caused maximum serologically detectable antibody levels to occur in the reserpine treated birds one day before maximum antibody levels in the gelatin group. The reserpine-treated birds exhibited maximum antibody levels five days after the antigen challenge as compared to six days in the gelatin group. These data represent a limited number of observations and only suggest that an earlier response occurred. Corticosterone Responses. Serum corticosterone response data for the seven treatment groups of Trials 1 and 2 are presented collectively in Table 2. The serum corticosterone levels of the birds that were given reserpine prior to SRBC were elevated significantly at the same time of antigen challenge as compared to the gelatin and saline groups. Serum corticosterone levels in reserpinetreated birds remained significantly elevated on day two, but these levels had fallen significantly below the levels in the gelatin and saline groups on day four and day six. However, on day eight no differences among the treatment groups were observed. No significant changes in the levels of serum corticosterone in the gelatin and saline groups were seen during the experimental period. The serum corticosterone responses of the
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on June 11, 2015
Days post-SRBC challenge 0 Treatment Reserpine (hours prior to antigen) 4.5 6.8 6.0 0.9 12 0 4.2 6.9 5.5 1.0 6 0 4.3 5.4 6.9 0.9 0 0 4.4 ± .4a 5.6 ± .2b 0.9 ± .2a 6.9 ± .2b Pooled 0 ± Oa Gelatin vehicle-control (hours prior to antigen) 5.5 4.4 0.5 12 0 5.9 5.6 0.5 3.9 6.1 6 0 5.0 0.4 5.8 4.1 0 0 5.4 ± .5a 4.1 ± .3a 5.9 ± .la 0.5 ± .2a Pooled 0 ± Oa Saline-control (hours prior to antigen) 4.5 ± .2a 4.8 ± .2a 0.6 ± .la 12 0 ± Oa 5.8 ± .2a 1 Pooled means with different small letters within a column are significantly different (P < .05).
1973
RESERPINE AND HUMORAL IMMUNITY
TABLE 2.—The mean1 (±S.E.) serum corticosterone levels (ng./ml.) in birds of Trials 1 and 2 Days post-SRBC Treatment
0
Reserpine (hours prior to antigen) 12 4.3 6 6.0 0 1.5
2
4
6
8
2.0 3.3 2.9
0.8 0.9 1.9
1.1 1.1 1.2
1.2±.2a
1.1 ± .2a
Gelatin vehicle-control (hours prior to antigen) 1.9 12 1.8 1.7 6 1.8 1.8 0 1.5 Pooled 1.7±.3a 1.8 ± .2a
1.7 1.6 1.9 1.7±.3b
2.0 1.7 1.6 1.8 ± .3b
1.8 1.7 1.7 1.7 ± .la
Saline-control (hours prior to antigen) 12 1.6±.4a
1.6±.2b
1.6 ± .4b
1.7 ± .5a
Pooled
1
3.9 ± .3b
1.8 ± .5a
Pooled means with different small letters within a column are significantly different (P < .05).
birds of Trial 3 are presented graphically in Figure 2. No significant changes in serum corticosterone levels of the gelatin group occurred during the experimental period. However, both reserpine treatments resulted in a significant, but transitory elevation of serum corticosterone. At the time of antigenic challenge serum corticosterone in birds given
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reserpine 6 hours earlier was significantly elevated as compared to both the gelatin control and the 0 hour reserpine-treated group. Both reserpine-treated groups showed significantly elevated levels through the third day after SRBC, but by the fourth day the serum corticosterone levels did not differ from the gelatin group. These results are consistent with those of Trials 1 and 2. The results of the three trials which depict the relationship of reserpine treatment to adrenal size and corticosterone concentration are presented in Table 3. Significant replicate differences or differences among treatment groups were not found. The reserpine treatments did not cause significant changes in relative total adrenal weights or in concentration of corticosterone in the adrenals.
-I L. 0 1 2 3 4 5 6 7 DAYS POST CHALLENGE WITH SRBC
FIG. 2. Serum corticosterone responses in chickens given either reserpine at 6 or 0 hours prior to the SRBC antigen challenge or the gelatin vehicle at 0 hours prior to the SRBC antigen challenge.
DISCUSSION Although the adrenocortical hormones have long been recognized as suppressants of humoral immunity in chickens (see Thaxton, 1971) and in mammals (Gisler and Schenkel-Hulliger, 1971), recent reports indicate that the adrenomedullary hormones, epinephrine and norepinephrine, also act as immunosuppressants in mammals (Melmon et al., 1974a, b; Bourne et al, 1974). The relationship of these adrenomedullary hor-
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on June 11, 2015
2.7 ± .2b
1.4 1.6 1.1 1.4 ± .3a
1974
E D E N S , SIKES, J R . , THAXTON AND MORGAN, J R .
TABLE 3.—Relative total adrenal weights and adrenal corticosterone by trial and treatment
mones, as well as that of reserpine, which is known to deplete the stores of these adrenomedullary hormones in chickens (Edens and Siegel, 1972; Ghosh and Datta, 1969), to immune responsiveness in the domestic fowl has not been reported previously. The data of the present report suggest that reserpine caused an enhancement of humoral immune responsiveness concomitant with increased adrenocortical activity. Maximum antibody levels in the reserpine-treated birds were significantly higher than those of the control birds. Additionally, maximum antibody levels in the reserpine-treated groups were found 24 hours before maximum levels in control groups. Furthermore, at the time of antigenic challenge and during the induction period of the primary response adrenocortical activity was stimulated as evidenced by significantly elevated serum corticosterone. At present definitive conclusions from the experimental data cannot be made. The results of this study suggest that some heretofore unrecognized interrelationship may exist between the adrenocortical and adrenomedullary secretions. It is possible that reserpine exerted direct or indirect affects on antibody production. Functionally, reserpine could have stimulated antibody synthesis directly by acting at the lymphoid-tissue level or it could have indirectly enhanced antibody production by causing a shift in the normal balance of the adrenomedullary cate-
cholamines and adrenocorticosteroids. Without additional information it would be fortuitous to support these or other explanations. However, it appears likely that both the adrenal cortex and medulla interact to influence immune responsiveness in the chicken.
REFERENCES Bourne, H. R., L. M. Kichtenstein, K. L. Melmon, C. S. Henny, Y. Weinstein and G. M. Shearer, 1974. Modulation of inflammation and immunity by cyclic AMP. Science, 184: 19-28. Edens, F. W., and H. S. Siegel, 1972. High ambient temperature and circulating catecholamines. Proc. Assn. South Agri. Workers, 69: 204. Edens, F. W., and H. S. Siegel, 1974a. Reserpine modification of the blood pH, pC0 2 , and p 0 2 of chickens in high environmental temperature. Poultry Sci. 53: 279-284. Edens, F. W., and H. S. Siegel, 1974b. Plasma corticosterone response in young chickens given sympatholytic agents and subjected to acute heat stress. Poultry Sci. 53: 1920. Ghosh, A., and B. Datta, 1969. Effect of reserpine on the release of adrenomedullary catecholamines and their role in glycemic and pressor responses in two avian species. Gen. Comp. Endocr. Suppl. 2: 354-357. Gisler, R. H., and L. Schenkel-Hulliger, 1971. Hormonal regulation of the immune response. II. Influence of pituitary and adrenal activity on immune responsiveness in vitro. Cell Immunol. 2: 646-657. Guillemin, R., G. W. Clayton, H. S. Lipscomb and J. O. Smith, 1959. Fluorometric measurement of rat plasma and adrenal corticosterone concentration. J. Lab. Clin. Med. 53: 830-832. Koelle, G. B., 1970. Neurohumoral transmission and
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on June 11, 2015
Adrenal corticosterone Adrenal weights (ng./100 mg. adrenal) (mg./lOOgm. body weight) Trial 3 Trial 3 Trials 1 and 2 Treatment Trials 1 and 2 3.28 ± .61a 14.2 ± .8a 2.32 ± .65a 16.0 ± .6a 1 Reserpine (80) (80) (58) (58) 2 3.65 ± .41a 13.5 ± .6a 2.40 ± .59a 15.1 ± .5a Vehicle (40) (40) (59) (59) 2.55 ± .54a 14.8 ± .8a Saline (20) (20) 1 Means ± SE with same small letters indicates no significant differences. 2 Numbers in parenthesis indicates number of birds per observation.
RESERPINE AND HUMORAL IMMUNITY
tein-binding radioassay. J. Clin. Endocr. 27: 973990. Ostle, B., 1963. Statistics in Research. Iowa State Univ. Press, Ames, Iowa. Srebocan, V., J. Pompe-Gotal and M. Plazonic, 1972. Investigations on the effect of stimulants on neurohumoral balance in chickens in intensive rearing. 5. Effects of ether methyl epireserpate (9064SU) on adrenocortical activity in stress. Vet. Archiv. 42: 71-76. SubbaRao, D. S. V.,and B. Glick, 1970. Immunosuppressive action of heat in chickens. Proc. Soc. Exp. Biol. Med. 133: 445-448. Thaxton, J. P., 1971. Immunodepression as affected by environmental temperature in chickens. Ph.D. Dissertation, Univ. Georgia, Athens, GA 30601. Thaxton, P., C. R. Sadler and B. Glick, 1968. The physiological response of New Hampshires to high temperatures. Poultry Sci. 46: 1598-1599. Thaxton, P., and H. S. Siegel, 1973. Modification of high temperature and ACTH induced immunodepression by metryapone. Poultry Sci. 52: 618-624. Witlin, B., 1967. Detection of antibodies by microtitration techniques. Mycopathol. et Mycologia Appl. 33: 241-257.
Efficacy of Turkey Herpesvirus Vaccine When Administered Simultaneously with Fowl Pox Vaccine C. S. ElDSON, P . VlLLEGAS ANDS. H . KLEVEN Poultry Disease Research Center, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30601 (Received for publication March 11, 1975)
ABSTRACT The efficacy of the turkey herpesvirus (HVT) vaccine in protecting chickens challenged with virulent Marek's disease (MD) virus was unaffected by the presence of either the chick embryo fowl pox vaccine or fowl pox vaccine derived from cell culture. Conversely the HVT vaccine did not affect the efficacy of the fowl pox vaccine in chickens challenged with pathogenic fowl pox virus. A combination of spectinomycin dihydrochloride pentahydrate and lincomycin hydrochloride monohydrate as well as spectinomycin sulfate tetrahydrate were found to be compatible with the HVT and fowl pox vaccines as demonstrated by resistance after challenge with virulent MD virus or fowl pox virus. POULTRY SCIENCE 54: 1975-1981, 1975
INTRODUCTION
M
AREK'S disease (MD) is world-wide in distribution and is responsible for considerable economic losses, particularly in areas of high density poultry production. Until recently MD cost the poultry industry
in the United States approximately 150-200 million dollars annually. Epidemiologic studies showed that MD is widespread among commercial flocks and under natural conditions most chickens become infected with MD virus at an early age (Witter et al., 1970a). The mode of transmission appears to be
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the autonomic nervous system. In: The Pharmacological Basis of Therapeutics. L. S. Goodman and A. Gilman, eds., 4th Ed. pp. 402-441. The McMillan Co., London-Toronto. Kramer, C. Y., 1956. Extension of multiple range tests to group means with unequal numbers of replications. Biometrics. 12: 307-310. Melmon, K. L., H. R. Bourne, Y. Weinstein, G. M. Shearer, S. Bauminger and J. Kramer, 1974a. Hemolytic plague formation by leukocytes in vitro: Control by vasoactive hormones. J. Clin. Invest. 53: 13-21. Melmon, K. L., Y. Weinstein, G. M. Shearer, H. R. Bourne and S. Bauminger, 1974b. Separation of specific antibody-forming mouse cells by their adherence to unsolubilized endogenous hormones. J. Clin. Invest. 53: 22-30. Montanari, R., and M. A. Stockham, 1962. Effects of single and repeated doses of reserpine on the secretion of adrenocorticotrophic hormone. Brit. J. Pharmacol. Chem. 18: 337-345. Murphy, B. E. P., 1967. Some studies of the proteinbinding of steroids and their applications to the routine micro and ultra-micro measurement of various steroids in body fluids by competitive pro-
1975
Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27607 (Received for publication March 11, 1975)
POULTRY SCIENCE 54: 1970-1975, 1975
INTRODUCTION
R
ESERPINE, which is used widely as an anti-hypertensive and tranquilizing agent, has been used as an anti-stress compound in chickens (see Edens and Siegel, 1974a). Reserpine acts at the level of the adrenal medulla and at post-ganglionic sympathetic nerve endings where the stores of the catecholamines are depleted by inhibition of uptake of dopamine and norepinephrine (Koelle, 1970). Additionally, reserpine is reported to cause a rise in the circulating levels of corticosterone in both mammals (Montanari and Stockham, 1962) and chickens (Srebocan et al., 1972; Edens and Siegel, 1974b). These reports suggest that this rise following reserpine treatment is the result of a concerted mobilization of hypophysial ACTH reserves. Exogenous ACTH is thought to suppress humoral immune responsiveness by stimulating the secretion of the adreno-
1. Paper Number 4606 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina. 2. A preliminary report of this paper was presented to the 63rd Annual Meeting of the Poultry Science Association, Morgantown, West Virginia. 3. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Experiment Station of the product named nor criticism of similar ones not mentioned.
cortical steriods (Thaxton et al., 1968; Subba Rao and Glick, 1970; Thaxton, 1971; Thaxton and Siegel, 1973). Melmon etal. (1974a, b) and Bourne et al. (1974) demonstrated that humoral antibody production is also suppressed by the catecholamines. Although both the catecholamines and adrenocortical steriods are reported to suppress humoral immune responses, their influence on immunity when considered collectively has not been reported. Thus, the objective of the present study was to determine the effects of reserpine on humoral immune responsiveness and to ascertain how the total adrenal may influence immune responsiveness in chickens. MATERIALS AND METHODS Cobb x Arbor Acre cockerels were used in three trials. The chicks were reared in heated brooding batteries until three weeks of age. Thereafter they were maintained in non-heated metal cages. A broiler startergrower ration and water were available ad libitum. In Trials 1 and 2 the chickens were randomized into seven treatment groups consisting of 10 chickens per group. When the chicks were four weeks of age reserpine, 4 4. Reserpine (3, 4, 5-trimethoxy-benzoyl methyl reserpate) obtained from Sigma Chemical Company, St. Louis, MO.
1970
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on June 11, 2015
ABSTRACT The role of reserpine in modifying a primary humoral immune response was evaluated in three experiments with young chickens. It was found that chickens injected with reserpine prior to an intravenous antigenic challenge with sheep red blood cells exhibited an enhanced primary humoral immune response. The enhanced response occurred concomitant with an elevation in adrenal cortical activity as evidenced by the significant elevation of serum corticosterone in the reserpine-treated chickens. These data suggest that both the adrenal cortex and medulla may influence immune responsiveness in the chicken.
1971
RESERPINE AND HUMORAL IMMUNITY
analysis of corticosterone at the following times: 1) immediately prior to the antigen challenge, 2) at 12 hour intervals for the first two days following the antigen and 3) daily thereafter for the next five days. Serum samples in all of the trials were collected according to the procedure described by Thaxton and Siegel (1973). The samples were assayed serologically by a microtitration procedure (Witlin, 1967) to determine the anti-SRBC antibody levels. Each sample was assayed by the competitive protein binding procedure of Murphy (1967) to determine the level of corticosterone. Nested analyses of variance were performed on all data according to Ostle (1963) and treatment differences were assigned by Kramer's (1956) modification of Duncan's multiple range test. Statements of significance are based on differences at the 5% level of probability. In Trials 1 and 2 no replicate differences were observed, therefore the data from these trials were pooled and are presented collectively. Trial 3 was of a different design and therefore it was analyzed and presented separately.
RESULTS Antibody Responses. The data illustrating the effects of reserpine on humoral immune responsiveness in the birds of Trials 1 and 2 are presented in Table 1. The mean antiSRBC antibody levels in birds receiving reserpine, regardless of time of administration, were higher than those of the gelatin or saline groups during the first six days of the primary hemagglutinin response. Antibody levels of the reserpine-treated birds were significantly higher than those of the gelatin and saline groups on both day four and day six. There were no significant differences among the treatment groups on day eight. These data indicate that the magnitude of the primary response was enhanced by the action of reserpine, but this enhancement appeared to
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on June 11, 2015
which was suspended in a 4% gelatin vehicle, was injected in a single intramuscular dose at the level of 0.75 mg./kg. of body weight. Three groups of birds were given reserpine at 12,6, or 0 hours prior to a single intravenous challenge with 1 ml. of a 10% saline suspension of sheep red blood cells (SRBC). Another three groups were designated as gelatin vehicle controls. They were injected intramuscularly with the 4% gelatin vehicle at the dose level of 1 ml. /kg. of body weight. These injections were given on the same schedule as the reserpine treatments. The final group of chickens, which served as saline controls, were given a single intramuscular injection of physiological saline at the level of 1 ml. /kg. of body weight. This injection was given 12 hours prior to the antigen. All the birds in Trials 1 and 2 were bled by venipuncture prior to the antigen challenge and thereafter at two day intervals for the next eight days to serologically monitor the ensuing primary hemagglutinin response. Following the final bleeding all the birds were weighed, killed by cervical dislocation, and the adrenal glands were excised and weighed. The adrenals were frozen (—20° C.) for subsequent analysis of corticosterone (Guillemin et al., 1959). In Trial 3, four week old cockerels were randomized into three groups of 40 birds each. Two groups were given reserpine (0.75 mg. /kg. of body weight) at 6 or 0 hours prior to antigen challenge with SRBC. The third or control group was injected with the gelatin vehicle at 0 hours prior to antigenic challenge. Differences between the saline controls and gelatin vehicle controls of Trials 1 and 2 were not observed, thus the saline controls were not used in Trial 3. To minimize the possibility of a handling stress from serial bleedings four birds, which were selected at random from each of the three groups, were used at each measurement interval. The birds were bled, killed, and the adrenals were collected for later chemical
1972
EDENS, SIKES, JR., THAXTON AND MORGAN, JR.
TABLE 1.—The mean> (±S.E.) anti-sheep red blood cell (SRBC) antibody levels (log2) for Trials 1 and 2
be transitory since there were no treatment differences at day eight. The antibody response data of the birds of Trial 3 are presented graphically in Figure 1. This trial was conducted to determine if reserpine shortened the response time for peak antibody titers. This result was suggested by the data of Trials 1 and 2. The results of Trial 3 indicate that the administration of reserpine prior to anti6 HOUR RESERPINE OHOUR RESERPINE OHOUR GELATIN
8
— —
hi > hi
>o o
6-
u
g2 CO
a
r
i
0 1 2 3 4 S 6 7 DAYS POST CHALLENGE WITH SRBC FIG. 1. Anti-SRBC antibody responses in chickens given either reserpine at 6 or 0 hours prior to the SRBC antigen challenge or the gelatin vehicle at 0 hours prior to the SRBC antigen challenge.
genie challenge caused maximum serologically detectable antibody levels to occur in the reserpine treated birds one day before maximum antibody levels in the gelatin group. The reserpine-treated birds exhibited maximum antibody levels five days after the antigen challenge as compared to six days in the gelatin group. These data represent a limited number of observations and only suggest that an earlier response occurred. Corticosterone Responses. Serum corticosterone response data for the seven treatment groups of Trials 1 and 2 are presented collectively in Table 2. The serum corticosterone levels of the birds that were given reserpine prior to SRBC were elevated significantly at the same time of antigen challenge as compared to the gelatin and saline groups. Serum corticosterone levels in reserpinetreated birds remained significantly elevated on day two, but these levels had fallen significantly below the levels in the gelatin and saline groups on day four and day six. However, on day eight no differences among the treatment groups were observed. No significant changes in the levels of serum corticosterone in the gelatin and saline groups were seen during the experimental period. The serum corticosterone responses of the
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on June 11, 2015
Days post-SRBC challenge 0 Treatment Reserpine (hours prior to antigen) 4.5 6.8 6.0 0.9 12 0 4.2 6.9 5.5 1.0 6 0 4.3 5.4 6.9 0.9 0 0 4.4 ± .4a 5.6 ± .2b 0.9 ± .2a 6.9 ± .2b Pooled 0 ± Oa Gelatin vehicle-control (hours prior to antigen) 5.5 4.4 0.5 12 0 5.9 5.6 0.5 3.9 6.1 6 0 5.0 0.4 5.8 4.1 0 0 5.4 ± .5a 4.1 ± .3a 5.9 ± .la 0.5 ± .2a Pooled 0 ± Oa Saline-control (hours prior to antigen) 4.5 ± .2a 4.8 ± .2a 0.6 ± .la 12 0 ± Oa 5.8 ± .2a 1 Pooled means with different small letters within a column are significantly different (P < .05).
1973
RESERPINE AND HUMORAL IMMUNITY
TABLE 2.—The mean1 (±S.E.) serum corticosterone levels (ng./ml.) in birds of Trials 1 and 2 Days post-SRBC Treatment
0
Reserpine (hours prior to antigen) 12 4.3 6 6.0 0 1.5
2
4
6
8
2.0 3.3 2.9
0.8 0.9 1.9
1.1 1.1 1.2
1.2±.2a
1.1 ± .2a
Gelatin vehicle-control (hours prior to antigen) 1.9 12 1.8 1.7 6 1.8 1.8 0 1.5 Pooled 1.7±.3a 1.8 ± .2a
1.7 1.6 1.9 1.7±.3b
2.0 1.7 1.6 1.8 ± .3b
1.8 1.7 1.7 1.7 ± .la
Saline-control (hours prior to antigen) 12 1.6±.4a
1.6±.2b
1.6 ± .4b
1.7 ± .5a
Pooled
1
3.9 ± .3b
1.8 ± .5a
Pooled means with different small letters within a column are significantly different (P < .05).
birds of Trial 3 are presented graphically in Figure 2. No significant changes in serum corticosterone levels of the gelatin group occurred during the experimental period. However, both reserpine treatments resulted in a significant, but transitory elevation of serum corticosterone. At the time of antigenic challenge serum corticosterone in birds given
io 5 o
J-l
6 HOUR RESERPINE . - - . 0HOUR RESERPINE • • - . OHOUR GELATIN ———•
§3
K UJ
82 § * p
Y &
Vt
^o—T
IE O
° I U W 0
reserpine 6 hours earlier was significantly elevated as compared to both the gelatin control and the 0 hour reserpine-treated group. Both reserpine-treated groups showed significantly elevated levels through the third day after SRBC, but by the fourth day the serum corticosterone levels did not differ from the gelatin group. These results are consistent with those of Trials 1 and 2. The results of the three trials which depict the relationship of reserpine treatment to adrenal size and corticosterone concentration are presented in Table 3. Significant replicate differences or differences among treatment groups were not found. The reserpine treatments did not cause significant changes in relative total adrenal weights or in concentration of corticosterone in the adrenals.
-I L. 0 1 2 3 4 5 6 7 DAYS POST CHALLENGE WITH SRBC
FIG. 2. Serum corticosterone responses in chickens given either reserpine at 6 or 0 hours prior to the SRBC antigen challenge or the gelatin vehicle at 0 hours prior to the SRBC antigen challenge.
DISCUSSION Although the adrenocortical hormones have long been recognized as suppressants of humoral immunity in chickens (see Thaxton, 1971) and in mammals (Gisler and Schenkel-Hulliger, 1971), recent reports indicate that the adrenomedullary hormones, epinephrine and norepinephrine, also act as immunosuppressants in mammals (Melmon et al., 1974a, b; Bourne et al, 1974). The relationship of these adrenomedullary hor-
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2.7 ± .2b
1.4 1.6 1.1 1.4 ± .3a
1974
E D E N S , SIKES, J R . , THAXTON AND MORGAN, J R .
TABLE 3.—Relative total adrenal weights and adrenal corticosterone by trial and treatment
mones, as well as that of reserpine, which is known to deplete the stores of these adrenomedullary hormones in chickens (Edens and Siegel, 1972; Ghosh and Datta, 1969), to immune responsiveness in the domestic fowl has not been reported previously. The data of the present report suggest that reserpine caused an enhancement of humoral immune responsiveness concomitant with increased adrenocortical activity. Maximum antibody levels in the reserpine-treated birds were significantly higher than those of the control birds. Additionally, maximum antibody levels in the reserpine-treated groups were found 24 hours before maximum levels in control groups. Furthermore, at the time of antigenic challenge and during the induction period of the primary response adrenocortical activity was stimulated as evidenced by significantly elevated serum corticosterone. At present definitive conclusions from the experimental data cannot be made. The results of this study suggest that some heretofore unrecognized interrelationship may exist between the adrenocortical and adrenomedullary secretions. It is possible that reserpine exerted direct or indirect affects on antibody production. Functionally, reserpine could have stimulated antibody synthesis directly by acting at the lymphoid-tissue level or it could have indirectly enhanced antibody production by causing a shift in the normal balance of the adrenomedullary cate-
cholamines and adrenocorticosteroids. Without additional information it would be fortuitous to support these or other explanations. However, it appears likely that both the adrenal cortex and medulla interact to influence immune responsiveness in the chicken.
REFERENCES Bourne, H. R., L. M. Kichtenstein, K. L. Melmon, C. S. Henny, Y. Weinstein and G. M. Shearer, 1974. Modulation of inflammation and immunity by cyclic AMP. Science, 184: 19-28. Edens, F. W., and H. S. Siegel, 1972. High ambient temperature and circulating catecholamines. Proc. Assn. South Agri. Workers, 69: 204. Edens, F. W., and H. S. Siegel, 1974a. Reserpine modification of the blood pH, pC0 2 , and p 0 2 of chickens in high environmental temperature. Poultry Sci. 53: 279-284. Edens, F. W., and H. S. Siegel, 1974b. Plasma corticosterone response in young chickens given sympatholytic agents and subjected to acute heat stress. Poultry Sci. 53: 1920. Ghosh, A., and B. Datta, 1969. Effect of reserpine on the release of adrenomedullary catecholamines and their role in glycemic and pressor responses in two avian species. Gen. Comp. Endocr. Suppl. 2: 354-357. Gisler, R. H., and L. Schenkel-Hulliger, 1971. Hormonal regulation of the immune response. II. Influence of pituitary and adrenal activity on immune responsiveness in vitro. Cell Immunol. 2: 646-657. Guillemin, R., G. W. Clayton, H. S. Lipscomb and J. O. Smith, 1959. Fluorometric measurement of rat plasma and adrenal corticosterone concentration. J. Lab. Clin. Med. 53: 830-832. Koelle, G. B., 1970. Neurohumoral transmission and
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Adrenal corticosterone Adrenal weights (ng./100 mg. adrenal) (mg./lOOgm. body weight) Trial 3 Trial 3 Trials 1 and 2 Treatment Trials 1 and 2 3.28 ± .61a 14.2 ± .8a 2.32 ± .65a 16.0 ± .6a 1 Reserpine (80) (80) (58) (58) 2 3.65 ± .41a 13.5 ± .6a 2.40 ± .59a 15.1 ± .5a Vehicle (40) (40) (59) (59) 2.55 ± .54a 14.8 ± .8a Saline (20) (20) 1 Means ± SE with same small letters indicates no significant differences. 2 Numbers in parenthesis indicates number of birds per observation.
RESERPINE AND HUMORAL IMMUNITY
tein-binding radioassay. J. Clin. Endocr. 27: 973990. Ostle, B., 1963. Statistics in Research. Iowa State Univ. Press, Ames, Iowa. Srebocan, V., J. Pompe-Gotal and M. Plazonic, 1972. Investigations on the effect of stimulants on neurohumoral balance in chickens in intensive rearing. 5. Effects of ether methyl epireserpate (9064SU) on adrenocortical activity in stress. Vet. Archiv. 42: 71-76. SubbaRao, D. S. V.,and B. Glick, 1970. Immunosuppressive action of heat in chickens. Proc. Soc. Exp. Biol. Med. 133: 445-448. Thaxton, J. P., 1971. Immunodepression as affected by environmental temperature in chickens. Ph.D. Dissertation, Univ. Georgia, Athens, GA 30601. Thaxton, P., C. R. Sadler and B. Glick, 1968. The physiological response of New Hampshires to high temperatures. Poultry Sci. 46: 1598-1599. Thaxton, P., and H. S. Siegel, 1973. Modification of high temperature and ACTH induced immunodepression by metryapone. Poultry Sci. 52: 618-624. Witlin, B., 1967. Detection of antibodies by microtitration techniques. Mycopathol. et Mycologia Appl. 33: 241-257.
Efficacy of Turkey Herpesvirus Vaccine When Administered Simultaneously with Fowl Pox Vaccine C. S. ElDSON, P . VlLLEGAS ANDS. H . KLEVEN Poultry Disease Research Center, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30601 (Received for publication March 11, 1975)
ABSTRACT The efficacy of the turkey herpesvirus (HVT) vaccine in protecting chickens challenged with virulent Marek's disease (MD) virus was unaffected by the presence of either the chick embryo fowl pox vaccine or fowl pox vaccine derived from cell culture. Conversely the HVT vaccine did not affect the efficacy of the fowl pox vaccine in chickens challenged with pathogenic fowl pox virus. A combination of spectinomycin dihydrochloride pentahydrate and lincomycin hydrochloride monohydrate as well as spectinomycin sulfate tetrahydrate were found to be compatible with the HVT and fowl pox vaccines as demonstrated by resistance after challenge with virulent MD virus or fowl pox virus. POULTRY SCIENCE 54: 1975-1981, 1975
INTRODUCTION
M
AREK'S disease (MD) is world-wide in distribution and is responsible for considerable economic losses, particularly in areas of high density poultry production. Until recently MD cost the poultry industry
in the United States approximately 150-200 million dollars annually. Epidemiologic studies showed that MD is widespread among commercial flocks and under natural conditions most chickens become infected with MD virus at an early age (Witter et al., 1970a). The mode of transmission appears to be
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the autonomic nervous system. In: The Pharmacological Basis of Therapeutics. L. S. Goodman and A. Gilman, eds., 4th Ed. pp. 402-441. The McMillan Co., London-Toronto. Kramer, C. Y., 1956. Extension of multiple range tests to group means with unequal numbers of replications. Biometrics. 12: 307-310. Melmon, K. L., H. R. Bourne, Y. Weinstein, G. M. Shearer, S. Bauminger and J. Kramer, 1974a. Hemolytic plague formation by leukocytes in vitro: Control by vasoactive hormones. J. Clin. Invest. 53: 13-21. Melmon, K. L., Y. Weinstein, G. M. Shearer, H. R. Bourne and S. Bauminger, 1974b. Separation of specific antibody-forming mouse cells by their adherence to unsolubilized endogenous hormones. J. Clin. Invest. 53: 22-30. Montanari, R., and M. A. Stockham, 1962. Effects of single and repeated doses of reserpine on the secretion of adrenocorticotrophic hormone. Brit. J. Pharmacol. Chem. 18: 337-345. Murphy, B. E. P., 1967. Some studies of the proteinbinding of steroids and their applications to the routine micro and ultra-micro measurement of various steroids in body fluids by competitive pro-
1975
