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Depression of Secondary Immunity by High Environmental Temperature1
Depression of Secondary Immunity by High Environmental Temperature 1 P . T H A X T O N 2 ' 3 AND H . S. S I E G E L 4
United States Department of Agriculture (Received for publication December 22, 1971)
POULTKY SCIENCE 51: 1519-1526,1972
INTRODUCTION
D
E V E L O P M E N T of primary immune responses in young chickens is suppressed b y high environmental temperature (Thaxton et at., 1968; Subba Rao and Glick, 1970). Hyperimmune agglutinin and precipitin responses were depressed within 12 hours following heating (Thaxton and Siegel, 1970), and it was suggested t h a t this immunodepression in chickens was a result of increased adrenal cortical hormone secretion. This study determined t h e effects of high environmental temperature on secondary agglutinin and precipitin responses in young chickens. I n addition, electrophoretic analysis of t h e circulating proteins was performed t o determine t h e effect of high temperature 1
A preliminary report of this paper was presented at the 67th Annual Meeting of the Association of Southern Agricultural Workers, Memphis, Tennessee, 1970. 2 Present address: Department of Poultry Science, North Carolina State University, Raleigh, N. C. 27607. 3 Portion of dissertation presented in partial fulfillment of requirements for Ph.D. at the University of Georgia. * Animal Science Research Division, A.R.S., U.S.D.A., Southeast Poultry Research Laboratory, 934 College Station Road, Athens, Georgia 30601.
immunodepression on the levels of specific serum proteins. MATERIALS AND METHODS Athens Randombred chickens from a flock developed and maintained a t t h e Southeast Poultry Research Laboratory, Athens, Georgia, were used in eight trials. T h e birds were subjected to four 30-minute exposures of 41.7° t o 43.3°C. on each of four consecutive hours. A t times other t h a n during t h e four heating exposures, the heat-treated birds along with the nonheated controls, were maintained a t temperatures of 23.9° to 25.0°C. T h e dewpoint temperature of both the heated and control environments was approximately 16.0° C. Feed and water were available ad libitum t o all birds during t h e experimental periods. Hemagglutination, using microtitration (Witlin, 1967) and precipitation using the capillary tube technique (Humphrey and White, 1964), were employed to serologically demonstrate antibody titers, respectively, to sheep red blood cells (S.R.B.C.) and bovine serum albumin (B.S.A.). Trials 1 through 4 were designed t o measure t h e immunodepressive effects of high temperature applied 24 hours after
1519
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ABSTRACT Young chickens, heated at temperatures of 41.7°-43.3°C. for 30 minute periods on the hour for four consecutive hours on the day following a secondary challenge with sheep red blood cells, produced lowered circulating agglutinins one day after heating but no effect was observed on the developing precipitin response to bovine serum albumin. When birds were similarly heated 24 hours after both the primary and secondary challenges with the aforementioned antigens, developing primary antibody levels were depressed at least two days after treatment in the sheep red blood cell-challenged birds and at five days post-heating in the bovine serum albumin-challenged birds. Depression of developing secondary antibody levels occurred only at the one-day post-heat bleeding in the sheep red blood cell-challenged birds. Immunodepression of secondary precipitin responses was not observed.
1520
P. T H A X T O N AND H. S. S I E G E L
the secondary antigenic challenge. F o r t y males were used in each trial; twenty were heated in the manner described previously and the remaining 20 served as nonheated controls. Trials 5 through 8 involved heating the birds 24 hours after both the primary and secondary antigenic challenges. In each of these trials 100 birds were used with 50 heated and the remaining 50 serving as controls.
Qualitative and quantitative studies of the serum proteins were made from serum samples taken from the birds in Trials 2, 4 and 7 which were then analyzed by acrylamide disc gel electrophoresis. Random samples of birds of a particular trial were made and the serum protein patterns of these same birds were determined at each bleeding period.
Glick (1968) found 17 bands plus two prealbumins in chicken serum using the above technique. He assigned the bands numbers beginning with band 17 which is found at the interface of the stacking and separating gels and ending with albumin as band 1. The two prealbumins were termed p 2 and pi. The stained bands, which were assigned a number after visual inspection of both the stained gel and the densitometric tracing, corresponded to those described by Glick (1968). Relative mobility values (Rm) of the bands were determined b y using the interface of the separating and stacking gels as the origin for calculation. The migration distance of each band from the origin was divided by the migration distance of band 10 (major transferrin) to determine the R m of each band, as described by Glick (1968) for the serum proteins of chickens and by Peacock et al. (1965) for those of humans. D a t a were evaluated by analysis of variance. Antibody titers were converted to
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The birds of Trials 1 through 4 were given primary antigen challenges, respectively, with S.R.B.C. (1 ml. of a 7 % saline suspension) or B.S.A. (30 m g . / K g . body weight) during the sixth week of age. T h e antigen injections were made intravenously via a brachial vein as were the bleedings for antisera testing. The primary immune response t h a t developed was monitored subsequently by bleeding and serological antibody determinations were made weekly for five consecutive weeks. Immediately after the fifth weekly bleeding the birds were challenged secondarily with their respective antigen at the same dose levels as those used for primary challenges. Twenty-four hours after this challenge half the birds in each of these four trials (Trials 1 through 4) were heat treated and all birds were then bled at two-day intervals for the following 10 days. Trials 5 through 8 were designed similarly to Trials 1 through 4 except t h a t heatings were conducted 24 hours after both primary and secondary challenges and bleedings were made a t two-day intervals for 10 days after both the primary and secondary antigen challenges.
Electrophoresis of the serum proteins was always made on sera taken within a previous 24-hour period. The procedure was t h a t described previously by Davis (1964) using a 7 % acrylamide solution to prepare the gels. T h e sample layer consisted of a 1:50 dilution of serum and acrylamide which was polymerized in a 7 X 6 3 mm. glass column. A tris buffer ( p H 8.4-8.6), containing 3.0 gm. of 2-amino-2-hydroxymethyl-3 propandiol (Tris) and 14.4 gm. of glycine in 1 liter of distilled water was used. Gels were stained for a t least one hour in a 7 % acetic acid solution saturated with aniline black and the excess stain was removed using a quick gel destainer apparatus (Canal Industrial Corp.). T h e various proteins were identified and quantitated densitometrically with a Photovolt densicord and integrator, using two microslits with a ratio of gel scan to recording paper speed of one to five.
H I G H TEMPERATURE
Primary Response
U First Artless 2ASs«eelAsti#ts IHHest
Primary Response
Secondary Response
Days After First Antigen
FIG. 1. The effect of high environmental temperature on the development of secondary immune responses in young chickens.
appropriate common logarithms to facilitate statistical analysis and, where appropriate, percentages were converted to arc sin V % - Comparisons of means were made using a modification of Duncan's new multiple-range test (Kramer, 1956). RESULTS
Depression oJ Secondary Responses. In Trials 1 through 4 serological measurements were made weekly after the primary antigenic stimulations to determine the rate of dissipation of the primary immune responses. This was performed to determine when a second antigen challenge should be given to effectively evoke secondary immune responses. In all four trials the greatest magnitudes of the primary agglutinin and precipitin responses occurred by seven days after the antigen challenge (Fig. 1). T h e antiS.R.B.C. levels of the birds of Trials 1 and 2 declined significantly at each succeeding seven-day sampling for 21 days. I n Trial 1 an additional decrease in anti-
body levels was also noted at the fourth bleeding (28 days). T h e anti-B.S.A. levels of the birds of Trials 3 and 4 were significantly depressed by 14 days after the first antigen and the additional decreases occurring during the last three periods were not significant. The data concerning high-temperature depression of the secondary responses of the birds of Trials 1 through 4 are also illustrated graphically in Figure 1. T h e only significant depression of secondary responses by heat in the birds of this segment of the study occurred in the S.R.B. C birds at day 37 of the secondary response, i.e., one day after heating; depression of secondary responses was not observed in B.S.A-birds. Trials 5 through 8 were conducted to ascertain the effects of exposure to high environmental temperature one day after both primary and secondary challenges with either S.R.B.C. or B.S.A. As shown in Figure 2, depression of primary responses was found in all four trials. T h e
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Non-Heated Heated PSO-05 #
Secondary Response
1521
IMMUNODEPRESSION
1522
P. THAXTON AND H. S. SIEGEL Secondary Respor
Days After First Antigen
lAindSA First an' Sacsni Antigiii lHlnd2H First ind Sscsnd Hiltinis
FIG. 2. The effect of high environmental tempeature on the development of primary and secondary immune responses in young chickens.
S.R.B.C.-challengedbirdsthatwereheated exhibited depressed antibody levels at four and two days after S.R.B.C.-challenge in Trials 5 and 6, respectively. Immunodepression also was demonstrated in the B.S.A.-challenged birds six days after the antigen in Trials 7 and 8. TABLE 1.—The > dative mobility values (Rm) of the serum proteins {Means + S.D.) of young chickens1'* Trials Bands 17 16 14 13 11 10 3 2 Alb pi Pi
3
4
7
0.19+.04 0.22+.03 0.36+.03 0.49+.03 0.8S+.02 1.00+.00 1.60+.06 1.93+.05 2.35+.09 2.60+.16 2.8S+.24
0.17+.03 0.22+.03 0.34+.05 0.48+.05 0.85+.03 1.00+.00 1.53+.05 1.83+.08 2.20+.07 2.43+.08 2.67+.12
0.18+.04 0.23+.04 0.34+.04 0.49+.03 0.83+.07 1.00+.00 1.53+ .13 1.87+ .11 2.22+.11 2.47+.11 2.70+.13
1 The Rm of each band was calculated by dividing the bands' migration distance from the origin by that of band 10. 2 Each mean represents the average mobility of the band over all time periods and includes the values of both the heated and non-heated birds.
Immunodepression of the secondary agglutinin responses occurred two days after S.R.B.C. challenge in the birds of Trial 5, but similar depression was not noted in the birds of Trial 6. Secondary precipitin responses to B.S.A. were not depressed by heating (Fig. 2). Serum Proteins During the Development of Primary and Secondary Immunity. In this study every bird analyzed by disc electrophoresis possessed the following bands as denned by Glick (1968): 17, 16, 14, 13, 11, 10, 3, 2, 1, p2, and pi. The Rm values of these bands are presented in Table 1. The small amount of variation in these values indicates the consistency of occurrence of these bands. All protein not included by one of the nine bands and two prealbumins was calculated as the residual fraction, i.e., the total of the various bands was subtracted from 100 to yield the residual percentage. A diagram representing a typical protein pattern as determined in this study is presented in Figure 3.
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Non-Heated Heated P < 0.05 *•
Primary Response
Secondary Response
Primary Response
1523
HIGH TEMPERATURE IMMUNODEPRESSION
w
PriMi
«... "
tHM*
FIG. 3. A typical protein pattern of the serum of young Athens Randombreds.
secondary S.R.B.C. challenge. The levels of band 13 were lowest one week after the primary challenge and highest at the next sampling. In these birds immuno-depression of the developing secondary agglutinin titers occurred two days after the P:i n.r , R. ! » • • • •
»
n
* 10
J—j, \
s •
a
5
C a CO
Y
6
\\
»
IE
4
2
0 A
2A
1H
~KST37—*—a—*3—ft ?A IK U F...t M(l| 2A$M»MAal
Days A f t e r First Antigen
XV
IH H i . l
FIG. 4. The effect of high environmental temperature on relative concentrations of the immunoglobulins of the chickens of Trial 2.
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No attempt was made to chemically identify the bands, since other workers have tentatively identified certain of these bands. Glick (1968) stated that IgM probably occurs in the region of bands 17 and 16 and that IgG is found similarly in the region of band 13. Bands 10 and 11 are known to be major transferrin and minor transferrin, respectively (Glick, 1968; Prosky et al., 1968). Band 1 is albumin and p2 and pi are the prealbumins (Glick, 1968; Prosky el al, 1968; Harris and Sweeney, 1969). Consistent changes occurred in the relative concentrations of bands 17, 16, 14 and 13, which are presumably the immunoglobulins. The analyses of the concentrations of these proteins are presented graphically in Figure 4, 5 and 6. The relative concentrations of bands 17, 16 and 14 of the birds of Trial 2 (Fig. 4) did not differ significantly at any of the five weekly sampling periods preceding the
1524
P. THAXTON AND H. S. SIEGEL
challenge resulted in fewer changes in the concentrations of the serum proteins of these birds than did a similar heat treatment after secondary S.R.B.C. challenge. The only notable effect was a decrease in the levels of band 17 two days after the antigen challenge. The birds of Trials 5 and 7 were treated with heat one day following the primary and secondary antigen challenges. Protein determinations were made only on the samples collected from the birds of Trial 7 (Fig. 6). The levels of band 13 in the heated birds were lowered at four days after the primary B.S.A. challenge and 10 days following the secondary B.S.A. challenge. The levels of band 16 were lower than controls two days after the first antigen, but at four days after the first antigen the levels of the heated birds were significantly higher than those of the controls. The only other significant change was that the levels of band 17 were in-
o. 5
~fl Nm-Hdlri Hiilad P*O.0S*
U Fittt Aitti|»» 2 * Sicond Anti|*i lHH«t
Days After First Antigen
FIG. 5. The effect of high environmental temperature on relative concentrations of the immunoglobulins of the chickens of Trial 4.
43
45
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antigen (see Fig. 1) and at this time (day 37) the relative concentrations of bands 14 and 13 were significantly lower than those of the non-heated controls. At the third sampling after heating, i.e., five days post-heating, the levels of band 14 were lower than the controls. Electrophoretic measurements were not made on samples collected from the birds of the replicate experiment, Trial 1. The protein analysis of the birds of Trial 4 (Fig. 5) that were administered a primary challenge with B.S.A. indicated that the relative concentrations of band 13 and 14 were not significantly different at any of the five consecutive weekly bleedings. Band 17 occurred at the highest level at the fifth bleeding and lowest at the third bleeding. The highest levels of band 16 were recorded one week after B.S.A. challenge and the lowest levels at the last bleeding. A single heat exposure one day following the secondary B.S.A.-
H I G H TEMPERATURE
IMMUNODEPRESSION
1525
laaaa2AfirttaaaSacaaa aaliaaai lNa«a2HHf«ta«aSacaa
Days After First Antigen
FIG. 6. The effect of high environmental temperature on relative concentrations of the immunoglobulins of the chickens of Trial 7.
creased two days following the second antigen. DISCUSSION Although depressions of both primary agglutinin (Trials 5 and 6) and precipitin responses (Trials 7 and 8) were observed, high temperature immunodepression of the developing secondary immune responses occurred only in the birds challenged with sheep red blood cells (Trials 1, 2, and 5) and not in the birds challenged with bovine serum albumin (Trials 3, 4, 7 and 8). In the chickens receiving primary immunization with S . R . B . C , heat treatment lowered the relative concentrations of the proteins comprising bands 13 and 14 (presumably IgG) at the same time t h a t immuno-depression occurred (Fig. 4). T h u s immunoglobulins synthesized during the induction phase of the primary and secondary agglutinin responses or their formation m a n y be more susceptible to the effects of high temperature than
those produced during the primary and secondary precipitin responses. Subba Rao and Glick (1970) reported the primary agglutinin response of young chickens to S.R.B.C. is sensitive to the effect of high temperature during the early p a r t of the induction phase. High-temperature immunodepression of both primary agglutinin and precipitin responses and the secondary agglutinin response, b u t not the secondary precipitin response, m a y be a result of a direct effect of high temperature on circulating immunoglobulins. Suppression of antiS.R.B.C. antibody formation has been reported to occur in mice possessing elevated serum calcium levels (Braun et al., 1970). Siegel and Latimer (1970) reported serum calcium levels to be increased by exogenous A C T H and Cortisol. The immunodepression noted in this experiment m a y therefore have resulted from increased levels of serum calcium or other circulating ions. Following temperature
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*r—a Nal-Hlitta Kaaiaa PS0.05*
1526
P. THAXTON AND H. S. SIEGEL
ACKNOWLEDGMENTS
We thank Mr. T. N. Lay for technical assistance. REFERENCES Braun, W., and M. Ishizuka, 1970. Suppression and enhancement of antibody formation by alteration of Ca 2+ levels. Nature, 226: 945-946. Davis, B. J., 1964. Disc electrophoresis. II. Methods and application to human serum proteins. Ann. New York Acad. Sci. 121: 404-428. Glick, B., 1968. Serum protein electrophoresis patterns in acrylamide gel: patterns from normal and bursaless birds. Poultry Sci. 47: 807-814.
Harris, G. C , Jr., and M. J. Sweeney, 1969. Electrophoretic evaluation of blood sera proteins of adult male chickens. Poultry Sci. 48: 1590-1592. Humphrey, J. H., and R. G. White, 1964. Immunology for Students of Medicine. F. A. Davis, Co., Philadelphia. Kramer, C. Y., 1956. Extension of multiple range tests to group means with unequal numbers of replications. Biometrics, 12: 307-310. Peacock, A. C , S. L. Bunting and K. G. Queen, 1965. Serum protein electrophoresis in acrylamide gel: patterns from normal human subjects. Science, 147: 1451-1453. Prosky, L., R. G. O'Dell, D. A. Libby and D. F. Flick, 1968. Lipoprotein staining following polyacrylamide disc electrophoresis of chick serum. Poultry Sci. 47: 185-189. Siegel, H. S., and J. W. Latimer, 1970. Bone and blood calcium responses to adrenocorticotropin, Cortisol and low environmental temperatures in young chickens. Proc. 14th World's Poultry Cong. Sec. I l l : 122-126. Sturkie, P. D., 1965. Avian Physiology, 2nd ed., Cornell Univ. Press, Ithaca, N. Y. Subba Rao, D. S. V., and B. Glick, 1970. Immunosuppressive action of heat in chickens. Proc. Soc. Exp. Biol. Med. 133: 445-448. Thaxton, P., C. R. Sadler and B. Glick, 1968. Immune response of chickens following heat exposure or injections with ACTH. Poultry Sci. 47: 264-266. Thaxton, P., and H. S. Siegel, 1970. Immunodepression in young chickens by high environmental temperature. Poultry Sci. 49: 202-205. Witlin, B., 1967. Detection of antibodies by microtitration techniques. Mycopathologia et Mycologia Applicata, 33: 241-257.
NEWS AND NOTES (Continued from page 1518} Ellis Cross has joined Pilch-DeKalb, Inc., Statesville, North Carolina, as Plant Manager of the North Carolina Hatchery Operations. He received B.S. and M.S. degrees at Auburn University.
partments of Pfizer Pharmaceuticals, and for systems support to Central Research and Quality Control.
PFIZER NOTES
A total of $40,000 in grants for animal health education was awarded by the Merck Company Foundation in 1971. The total equals the amount awarded in 1970 to further education in animal science. Recipients of grants last year included:
Dr. G. M. K. Hughes has been appointed Vice President-Administration of Pfizer Pharmaceuticals, a major organizational unit of Pfitzer, Inc. He will be responsible for the Systems, Planning, Human Resources and Sales Operations De-
MERCK NOTES
(Continued on page 1577)
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treatment certain physical characteristics of the immunoglobulin molecules may be altered so that a loss in reactive specificity of the antibodies for their respective antigens occurs rather than a reduction in the circulating levels of immunoglobulins. Increased water consumption and subsequent hemodilution (Sturkie, 1965) does not explain these results since they occur the day after the heat exposure and they are limited to specific proteins, presumably IgG. Studies are needed to determine if high-temperature immunodepression is caused by specific changes in circulating antibody molecules or as a result of interrupted cellular synthesis of proteins. These results do not rule out the possibility that increased secretion of adrenal steroids are involved.
United States Department of Agriculture (Received for publication December 22, 1971)
POULTKY SCIENCE 51: 1519-1526,1972
INTRODUCTION
D
E V E L O P M E N T of primary immune responses in young chickens is suppressed b y high environmental temperature (Thaxton et at., 1968; Subba Rao and Glick, 1970). Hyperimmune agglutinin and precipitin responses were depressed within 12 hours following heating (Thaxton and Siegel, 1970), and it was suggested t h a t this immunodepression in chickens was a result of increased adrenal cortical hormone secretion. This study determined t h e effects of high environmental temperature on secondary agglutinin and precipitin responses in young chickens. I n addition, electrophoretic analysis of t h e circulating proteins was performed t o determine t h e effect of high temperature 1
A preliminary report of this paper was presented at the 67th Annual Meeting of the Association of Southern Agricultural Workers, Memphis, Tennessee, 1970. 2 Present address: Department of Poultry Science, North Carolina State University, Raleigh, N. C. 27607. 3 Portion of dissertation presented in partial fulfillment of requirements for Ph.D. at the University of Georgia. * Animal Science Research Division, A.R.S., U.S.D.A., Southeast Poultry Research Laboratory, 934 College Station Road, Athens, Georgia 30601.
immunodepression on the levels of specific serum proteins. MATERIALS AND METHODS Athens Randombred chickens from a flock developed and maintained a t t h e Southeast Poultry Research Laboratory, Athens, Georgia, were used in eight trials. T h e birds were subjected to four 30-minute exposures of 41.7° t o 43.3°C. on each of four consecutive hours. A t times other t h a n during t h e four heating exposures, the heat-treated birds along with the nonheated controls, were maintained a t temperatures of 23.9° to 25.0°C. T h e dewpoint temperature of both the heated and control environments was approximately 16.0° C. Feed and water were available ad libitum t o all birds during t h e experimental periods. Hemagglutination, using microtitration (Witlin, 1967) and precipitation using the capillary tube technique (Humphrey and White, 1964), were employed to serologically demonstrate antibody titers, respectively, to sheep red blood cells (S.R.B.C.) and bovine serum albumin (B.S.A.). Trials 1 through 4 were designed t o measure t h e immunodepressive effects of high temperature applied 24 hours after
1519
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ABSTRACT Young chickens, heated at temperatures of 41.7°-43.3°C. for 30 minute periods on the hour for four consecutive hours on the day following a secondary challenge with sheep red blood cells, produced lowered circulating agglutinins one day after heating but no effect was observed on the developing precipitin response to bovine serum albumin. When birds were similarly heated 24 hours after both the primary and secondary challenges with the aforementioned antigens, developing primary antibody levels were depressed at least two days after treatment in the sheep red blood cell-challenged birds and at five days post-heating in the bovine serum albumin-challenged birds. Depression of developing secondary antibody levels occurred only at the one-day post-heat bleeding in the sheep red blood cell-challenged birds. Immunodepression of secondary precipitin responses was not observed.
1520
P. T H A X T O N AND H. S. S I E G E L
the secondary antigenic challenge. F o r t y males were used in each trial; twenty were heated in the manner described previously and the remaining 20 served as nonheated controls. Trials 5 through 8 involved heating the birds 24 hours after both the primary and secondary antigenic challenges. In each of these trials 100 birds were used with 50 heated and the remaining 50 serving as controls.
Qualitative and quantitative studies of the serum proteins were made from serum samples taken from the birds in Trials 2, 4 and 7 which were then analyzed by acrylamide disc gel electrophoresis. Random samples of birds of a particular trial were made and the serum protein patterns of these same birds were determined at each bleeding period.
Glick (1968) found 17 bands plus two prealbumins in chicken serum using the above technique. He assigned the bands numbers beginning with band 17 which is found at the interface of the stacking and separating gels and ending with albumin as band 1. The two prealbumins were termed p 2 and pi. The stained bands, which were assigned a number after visual inspection of both the stained gel and the densitometric tracing, corresponded to those described by Glick (1968). Relative mobility values (Rm) of the bands were determined b y using the interface of the separating and stacking gels as the origin for calculation. The migration distance of each band from the origin was divided by the migration distance of band 10 (major transferrin) to determine the R m of each band, as described by Glick (1968) for the serum proteins of chickens and by Peacock et al. (1965) for those of humans. D a t a were evaluated by analysis of variance. Antibody titers were converted to
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on May 26, 2015
The birds of Trials 1 through 4 were given primary antigen challenges, respectively, with S.R.B.C. (1 ml. of a 7 % saline suspension) or B.S.A. (30 m g . / K g . body weight) during the sixth week of age. T h e antigen injections were made intravenously via a brachial vein as were the bleedings for antisera testing. The primary immune response t h a t developed was monitored subsequently by bleeding and serological antibody determinations were made weekly for five consecutive weeks. Immediately after the fifth weekly bleeding the birds were challenged secondarily with their respective antigen at the same dose levels as those used for primary challenges. Twenty-four hours after this challenge half the birds in each of these four trials (Trials 1 through 4) were heat treated and all birds were then bled at two-day intervals for the following 10 days. Trials 5 through 8 were designed similarly to Trials 1 through 4 except t h a t heatings were conducted 24 hours after both primary and secondary challenges and bleedings were made a t two-day intervals for 10 days after both the primary and secondary antigen challenges.
Electrophoresis of the serum proteins was always made on sera taken within a previous 24-hour period. The procedure was t h a t described previously by Davis (1964) using a 7 % acrylamide solution to prepare the gels. T h e sample layer consisted of a 1:50 dilution of serum and acrylamide which was polymerized in a 7 X 6 3 mm. glass column. A tris buffer ( p H 8.4-8.6), containing 3.0 gm. of 2-amino-2-hydroxymethyl-3 propandiol (Tris) and 14.4 gm. of glycine in 1 liter of distilled water was used. Gels were stained for a t least one hour in a 7 % acetic acid solution saturated with aniline black and the excess stain was removed using a quick gel destainer apparatus (Canal Industrial Corp.). T h e various proteins were identified and quantitated densitometrically with a Photovolt densicord and integrator, using two microslits with a ratio of gel scan to recording paper speed of one to five.
H I G H TEMPERATURE
Primary Response
U First Artless 2ASs«eelAsti#ts IHHest
Primary Response
Secondary Response
Days After First Antigen
FIG. 1. The effect of high environmental temperature on the development of secondary immune responses in young chickens.
appropriate common logarithms to facilitate statistical analysis and, where appropriate, percentages were converted to arc sin V % - Comparisons of means were made using a modification of Duncan's new multiple-range test (Kramer, 1956). RESULTS
Depression oJ Secondary Responses. In Trials 1 through 4 serological measurements were made weekly after the primary antigenic stimulations to determine the rate of dissipation of the primary immune responses. This was performed to determine when a second antigen challenge should be given to effectively evoke secondary immune responses. In all four trials the greatest magnitudes of the primary agglutinin and precipitin responses occurred by seven days after the antigen challenge (Fig. 1). T h e antiS.R.B.C. levels of the birds of Trials 1 and 2 declined significantly at each succeeding seven-day sampling for 21 days. I n Trial 1 an additional decrease in anti-
body levels was also noted at the fourth bleeding (28 days). T h e anti-B.S.A. levels of the birds of Trials 3 and 4 were significantly depressed by 14 days after the first antigen and the additional decreases occurring during the last three periods were not significant. The data concerning high-temperature depression of the secondary responses of the birds of Trials 1 through 4 are also illustrated graphically in Figure 1. T h e only significant depression of secondary responses by heat in the birds of this segment of the study occurred in the S.R.B. C birds at day 37 of the secondary response, i.e., one day after heating; depression of secondary responses was not observed in B.S.A-birds. Trials 5 through 8 were conducted to ascertain the effects of exposure to high environmental temperature one day after both primary and secondary challenges with either S.R.B.C. or B.S.A. As shown in Figure 2, depression of primary responses was found in all four trials. T h e
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Non-Heated Heated PSO-05 #
Secondary Response
1521
IMMUNODEPRESSION
1522
P. THAXTON AND H. S. SIEGEL Secondary Respor
Days After First Antigen
lAindSA First an' Sacsni Antigiii lHlnd2H First ind Sscsnd Hiltinis
FIG. 2. The effect of high environmental tempeature on the development of primary and secondary immune responses in young chickens.
S.R.B.C.-challengedbirdsthatwereheated exhibited depressed antibody levels at four and two days after S.R.B.C.-challenge in Trials 5 and 6, respectively. Immunodepression also was demonstrated in the B.S.A.-challenged birds six days after the antigen in Trials 7 and 8. TABLE 1.—The > dative mobility values (Rm) of the serum proteins {Means + S.D.) of young chickens1'* Trials Bands 17 16 14 13 11 10 3 2 Alb pi Pi
3
4
7
0.19+.04 0.22+.03 0.36+.03 0.49+.03 0.8S+.02 1.00+.00 1.60+.06 1.93+.05 2.35+.09 2.60+.16 2.8S+.24
0.17+.03 0.22+.03 0.34+.05 0.48+.05 0.85+.03 1.00+.00 1.53+.05 1.83+.08 2.20+.07 2.43+.08 2.67+.12
0.18+.04 0.23+.04 0.34+.04 0.49+.03 0.83+.07 1.00+.00 1.53+ .13 1.87+ .11 2.22+.11 2.47+.11 2.70+.13
1 The Rm of each band was calculated by dividing the bands' migration distance from the origin by that of band 10. 2 Each mean represents the average mobility of the band over all time periods and includes the values of both the heated and non-heated birds.
Immunodepression of the secondary agglutinin responses occurred two days after S.R.B.C. challenge in the birds of Trial 5, but similar depression was not noted in the birds of Trial 6. Secondary precipitin responses to B.S.A. were not depressed by heating (Fig. 2). Serum Proteins During the Development of Primary and Secondary Immunity. In this study every bird analyzed by disc electrophoresis possessed the following bands as denned by Glick (1968): 17, 16, 14, 13, 11, 10, 3, 2, 1, p2, and pi. The Rm values of these bands are presented in Table 1. The small amount of variation in these values indicates the consistency of occurrence of these bands. All protein not included by one of the nine bands and two prealbumins was calculated as the residual fraction, i.e., the total of the various bands was subtracted from 100 to yield the residual percentage. A diagram representing a typical protein pattern as determined in this study is presented in Figure 3.
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Primary Response
Secondary Response
Primary Response
1523
HIGH TEMPERATURE IMMUNODEPRESSION
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FIG. 3. A typical protein pattern of the serum of young Athens Randombreds.
secondary S.R.B.C. challenge. The levels of band 13 were lowest one week after the primary challenge and highest at the next sampling. In these birds immuno-depression of the developing secondary agglutinin titers occurred two days after the P:i n.r , R. ! » • • • •
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FIG. 4. The effect of high environmental temperature on relative concentrations of the immunoglobulins of the chickens of Trial 2.
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No attempt was made to chemically identify the bands, since other workers have tentatively identified certain of these bands. Glick (1968) stated that IgM probably occurs in the region of bands 17 and 16 and that IgG is found similarly in the region of band 13. Bands 10 and 11 are known to be major transferrin and minor transferrin, respectively (Glick, 1968; Prosky et al., 1968). Band 1 is albumin and p2 and pi are the prealbumins (Glick, 1968; Prosky el al, 1968; Harris and Sweeney, 1969). Consistent changes occurred in the relative concentrations of bands 17, 16, 14 and 13, which are presumably the immunoglobulins. The analyses of the concentrations of these proteins are presented graphically in Figure 4, 5 and 6. The relative concentrations of bands 17, 16 and 14 of the birds of Trial 2 (Fig. 4) did not differ significantly at any of the five weekly sampling periods preceding the
1524
P. THAXTON AND H. S. SIEGEL
challenge resulted in fewer changes in the concentrations of the serum proteins of these birds than did a similar heat treatment after secondary S.R.B.C. challenge. The only notable effect was a decrease in the levels of band 17 two days after the antigen challenge. The birds of Trials 5 and 7 were treated with heat one day following the primary and secondary antigen challenges. Protein determinations were made only on the samples collected from the birds of Trial 7 (Fig. 6). The levels of band 13 in the heated birds were lowered at four days after the primary B.S.A. challenge and 10 days following the secondary B.S.A. challenge. The levels of band 16 were lower than controls two days after the first antigen, but at four days after the first antigen the levels of the heated birds were significantly higher than those of the controls. The only other significant change was that the levels of band 17 were in-
o. 5
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FIG. 5. The effect of high environmental temperature on relative concentrations of the immunoglobulins of the chickens of Trial 4.
43
45
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antigen (see Fig. 1) and at this time (day 37) the relative concentrations of bands 14 and 13 were significantly lower than those of the non-heated controls. At the third sampling after heating, i.e., five days post-heating, the levels of band 14 were lower than the controls. Electrophoretic measurements were not made on samples collected from the birds of the replicate experiment, Trial 1. The protein analysis of the birds of Trial 4 (Fig. 5) that were administered a primary challenge with B.S.A. indicated that the relative concentrations of band 13 and 14 were not significantly different at any of the five consecutive weekly bleedings. Band 17 occurred at the highest level at the fifth bleeding and lowest at the third bleeding. The highest levels of band 16 were recorded one week after B.S.A. challenge and the lowest levels at the last bleeding. A single heat exposure one day following the secondary B.S.A.-
H I G H TEMPERATURE
IMMUNODEPRESSION
1525
laaaa2AfirttaaaSacaaa aaliaaai lNa«a2HHf«ta«aSacaa
Days After First Antigen
FIG. 6. The effect of high environmental temperature on relative concentrations of the immunoglobulins of the chickens of Trial 7.
creased two days following the second antigen. DISCUSSION Although depressions of both primary agglutinin (Trials 5 and 6) and precipitin responses (Trials 7 and 8) were observed, high temperature immunodepression of the developing secondary immune responses occurred only in the birds challenged with sheep red blood cells (Trials 1, 2, and 5) and not in the birds challenged with bovine serum albumin (Trials 3, 4, 7 and 8). In the chickens receiving primary immunization with S . R . B . C , heat treatment lowered the relative concentrations of the proteins comprising bands 13 and 14 (presumably IgG) at the same time t h a t immuno-depression occurred (Fig. 4). T h u s immunoglobulins synthesized during the induction phase of the primary and secondary agglutinin responses or their formation m a n y be more susceptible to the effects of high temperature than
those produced during the primary and secondary precipitin responses. Subba Rao and Glick (1970) reported the primary agglutinin response of young chickens to S.R.B.C. is sensitive to the effect of high temperature during the early p a r t of the induction phase. High-temperature immunodepression of both primary agglutinin and precipitin responses and the secondary agglutinin response, b u t not the secondary precipitin response, m a y be a result of a direct effect of high temperature on circulating immunoglobulins. Suppression of antiS.R.B.C. antibody formation has been reported to occur in mice possessing elevated serum calcium levels (Braun et al., 1970). Siegel and Latimer (1970) reported serum calcium levels to be increased by exogenous A C T H and Cortisol. The immunodepression noted in this experiment m a y therefore have resulted from increased levels of serum calcium or other circulating ions. Following temperature
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1526
P. THAXTON AND H. S. SIEGEL
ACKNOWLEDGMENTS
We thank Mr. T. N. Lay for technical assistance. REFERENCES Braun, W., and M. Ishizuka, 1970. Suppression and enhancement of antibody formation by alteration of Ca 2+ levels. Nature, 226: 945-946. Davis, B. J., 1964. Disc electrophoresis. II. Methods and application to human serum proteins. Ann. New York Acad. Sci. 121: 404-428. Glick, B., 1968. Serum protein electrophoresis patterns in acrylamide gel: patterns from normal and bursaless birds. Poultry Sci. 47: 807-814.
Harris, G. C , Jr., and M. J. Sweeney, 1969. Electrophoretic evaluation of blood sera proteins of adult male chickens. Poultry Sci. 48: 1590-1592. Humphrey, J. H., and R. G. White, 1964. Immunology for Students of Medicine. F. A. Davis, Co., Philadelphia. Kramer, C. Y., 1956. Extension of multiple range tests to group means with unequal numbers of replications. Biometrics, 12: 307-310. Peacock, A. C , S. L. Bunting and K. G. Queen, 1965. Serum protein electrophoresis in acrylamide gel: patterns from normal human subjects. Science, 147: 1451-1453. Prosky, L., R. G. O'Dell, D. A. Libby and D. F. Flick, 1968. Lipoprotein staining following polyacrylamide disc electrophoresis of chick serum. Poultry Sci. 47: 185-189. Siegel, H. S., and J. W. Latimer, 1970. Bone and blood calcium responses to adrenocorticotropin, Cortisol and low environmental temperatures in young chickens. Proc. 14th World's Poultry Cong. Sec. I l l : 122-126. Sturkie, P. D., 1965. Avian Physiology, 2nd ed., Cornell Univ. Press, Ithaca, N. Y. Subba Rao, D. S. V., and B. Glick, 1970. Immunosuppressive action of heat in chickens. Proc. Soc. Exp. Biol. Med. 133: 445-448. Thaxton, P., C. R. Sadler and B. Glick, 1968. Immune response of chickens following heat exposure or injections with ACTH. Poultry Sci. 47: 264-266. Thaxton, P., and H. S. Siegel, 1970. Immunodepression in young chickens by high environmental temperature. Poultry Sci. 49: 202-205. Witlin, B., 1967. Detection of antibodies by microtitration techniques. Mycopathologia et Mycologia Applicata, 33: 241-257.
NEWS AND NOTES (Continued from page 1518} Ellis Cross has joined Pilch-DeKalb, Inc., Statesville, North Carolina, as Plant Manager of the North Carolina Hatchery Operations. He received B.S. and M.S. degrees at Auburn University.
partments of Pfizer Pharmaceuticals, and for systems support to Central Research and Quality Control.
PFIZER NOTES
A total of $40,000 in grants for animal health education was awarded by the Merck Company Foundation in 1971. The total equals the amount awarded in 1970 to further education in animal science. Recipients of grants last year included:
Dr. G. M. K. Hughes has been appointed Vice President-Administration of Pfizer Pharmaceuticals, a major organizational unit of Pfitzer, Inc. He will be responsible for the Systems, Planning, Human Resources and Sales Operations De-
MERCK NOTES
(Continued on page 1577)
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treatment certain physical characteristics of the immunoglobulin molecules may be altered so that a loss in reactive specificity of the antibodies for their respective antigens occurs rather than a reduction in the circulating levels of immunoglobulins. Increased water consumption and subsequent hemodilution (Sturkie, 1965) does not explain these results since they occur the day after the heat exposure and they are limited to specific proteins, presumably IgG. Studies are needed to determine if high-temperature immunodepression is caused by specific changes in circulating antibody molecules or as a result of interrupted cellular synthesis of proteins. These results do not rule out the possibility that increased secretion of adrenal steroids are involved.