Effect of Cold Exposure on The Immune Response of Chickens D. S. V. SUBBA RAO and B. GLICK1 Department of Poultry Science, Mississippi Agricultural & Forestry Experiment Station, Mississippi State University, Mississippi State, Mississippi 39762 (Received for publication October 25, 1976)
Poultry Science 56:992-996, 1977 INTRODUCTION
Considerable research has been reported on changes in the physiology of domestic fowls exposed to stressful environmental temperatures. In all studies measurements of adrenal activity is frequently used as a criterion for physiological stress (Fortier, 1959; Fortier and DeGroot, 1959). The control of antibody-mediated immunity by varying environmental temperatures has been central to the research of our laboratory. Initial investigations in our laboratory identified the suppressive influence of acute heat on antibody production (Thaxton et al, 1968; Subba Rao and Glick, 1970). The natural exposure of birds to chronic periods of heat (or cold) occurs in many regions of the world. This prompted us to extend our immune studies to more prolonged periods of heat exposure. A search of the literature revealed a report of reduced infection to pasteurella in the presence of low temperature (Juszkiewicz, 1967). We included in our immune studies the exposure of birds to chronic and acute cold. MATERIALS AND METHODS Birds: Birds were from a commercial broiler flock or from a closed flock of New Hampshire, an American breed, maintained by Prof. L. J.
1
Please send all correspondence to: Bruce Glick, Ph.D., Professor, Poultry Science Department, P.O. Box 5188, Mississippi State University, Mississippi State, MS 39762.
Dreesen of our Poultry Department. Birds were reared either in all metal batteries placed in environmental chambers or on the floor of the environmental chambers with wood shavings as litter. The control birds were reared at ambient temperature. Environmental chambers: The environmental chambers were maintained by the South C e n t r a l Research Laboratory (U.S.D.A., A.R.S.). Each chamber (6.08 m. 2 floor area) was designed and built by Reece and Deaton (Reece and Deaton, 1967). The air exchange rate was 94.61/sec./chamber, which reduced ammonia, dust and carbon dioxide to a negligible factor, since ventilation was composed entirely of fresh air. The temperatures in the chambers were established at 7.2 C , 24 C , 32.2°C., 40.6°C. and 7.2°C.-32.2 C C. cycle over a 12 hour period with dewpoint temperature of 0°C. in 7.2°C. chamber and 10°C. in all other chambers. These constant conditions were maintained within ± 1 C. The light intensity for all treatments was an average of 1.2 foot candles at bird level for the first 3 weeks and 0.75 foot candles for the remaining time of the experimental period. Cold or Heat treatments: In the first set of experiments chickens were subjected to chronic cold or heat treatments from the day of hatch to 4 weeks of age in environmental chambers set at 7.2°C, 24°C, 7.2°C.-32.2°C. and 40.6 C. (Figure 1). In the second series of experiments the chickens were exposed to 30 minute periods of cold each hour for 2 or 4
992
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 31, 2015
ABSTRACT The effect of various environmental temperature changes on the antibody production of chickens was examined. Chronic cold exposure significantly increased the antibody titers. Birds exposed to 32.2°C. and above had significantly depressed agglutinin levels. Short term cold exposures 2 or 4 times following the antigen injection enhanced the agglutinin and hemolysin response. Thirty minute cold exposures for 2 or 4 times significantly increased IgM antibody production and markedly reduced the IgG antibody. The data suggest the elevation of antibody titers are in relation to time of the cold treatment and antigen injection. Observed differences of antibody production may be due to cold induced changes in the metabolic activity of antigen reactive cells and antibody producing cells.
993
COLD AND IMMUNE RESPONSE
— 7.2°c ••• 24°c — Control -»7.2°c-32.2°c — 32 2°c — 40.6°c
1 3
4
5
6
7
8
9
10
II
12
FIG. 1. Antibody titers of birds exposed to various environmental temperatures.
consecutive hours at the time of antigen injection (Figure 2). In the third series of experiments the birds were subjected to 30-minute periods of cold (Figure 4) either 12 hours before [-12, - 1 1 , - 1 0 , - 9 ] or 12 hours after [+12, +13, +15 hours] antigen administration. Antigens: The antigens used were sheep red blood cells (SRBC) and bovine serum albumin (BSA). SRBC stored in Alsever's solution were washed 3 times in physiological saline (0.9% NaCl) and resuspended to a 7 percent suspension. BSA was dissolved in saline to give a concentration of 40 mg./ml. Immunizations: All immunizations were made intravenously either with 1 ml. of the 7 percent suspension of SRBC or with BSA at the rate of 40 mg./kg. of body weight. In all experiments the type of antigen, the dosage of antigen, administration and vehicle, the time and methods of assessment of immune response were kept as constant as possible.
TOTAL ANTIBODY TITERS
ME-SENSITIVE ANTIBODY TITERS
DAYS AFTER
SRBC
ME-RESISTANT ANTIBODY TITERS
INJECTION
RESULTS FIG. 2. Antibody titers of birds exposed to short term cold treatments (chickens exposed to 30-minute periods of cold; CS-2, two times; CS-4, four times).
Chronic cold (7.2°C.) exposure significantly increased the antibody titers. Exposure to
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 31, 2015
2
DAYS AFTER SRBC INJECTION
Hemagglutinin assay: A microtiter procedure was employed for antibody titers (Wegman and Smithies, 1966). The birds were bled from the brachial vein 3 or 4 times between the 3rd and 10th day after immunization to collect serum for the micromethod. Serum samples were titrated individually (Subba Rao and Glick, 1975). Titers were expressed as the log 2 of the reciprocal of the highest dilutions giving visible agglutination. 2-Mercaptoethanol (ME) sensitive and resistant antibody: Equal volumes of serum and 0.2 m. ME in phosphate buffered saline (pH 7.4) were mixed and incubated at 37 C. for 30 minutes prior to serial dilution (Delhanty and Soloman, 1966). Agglutination tests were conducted and the titer was recorded as ME-resistant antibody. The reduction of the titer due to ME treatment was recorded as ME sensitive antibody. Hemolysin response: The hemolysin titers were determined using the microtiter procedure (Wegman and Smithies, 1966; Takatsy and Hamar, 1955). Serum dilutions were carried out in modified barbital buffer (Mayer et al., 1946). After the addition of SRBC, 25 jul. of complement was added to all the wells and the reaction mixture was incubated at 37 C. for 30 minutes. Titers were expressed as the log 2 like agglutinin levels. BSA precipitin levels: Birds were bled 7 days after the immunization. The antibody nitrogen (jug. AbN/ml.) in the serum was determined using a quantitative precepitin test (May and Glick, 1964). Plaque Forming Cell (PFC) assay: An agarless monolayer hemolytic plaque technique (Cunningham and Szenberg, 1968) as modified in our laboratory for the chicken system (Mueller et al., 1971) was employed. Four or five days following an I.V. injection of 1 ml. of a 7 percent suspension of SRBC, spleens were removed and antibody producing cells counted by the plaque forming assay (Subba Rao and Glick, 1975). The results were expressed as the number of PFC per 10 6 spleen cells. Statistical analysis: All the data were analyzed by analysis of variance (Steel and Torrie, 1960). Significant differences were determined by Duncan's new multiple range test (Duncan, 1955).
SUBBA RAO AND GLICK
994
DISCUSSION
DAYS AFTER SRBC INJECTION
FIG. 3. Hemolysin response of birds exposed to short term cold treatments (chickens exposed to 30-minute periods of cold; CS-2, two times; CS-4, four times). 24 C. increased the agglutinin levels but not significantly. Birds exposed to 32.2 C. and above experienced a significant suppression of their agglutinin levels (Figure 1). Birds exposed to cold 2 or 4 times immediately after the injection had significantly higher agglutinin and hemolysin responses than controls (Figures 2 and 3). The thirty minute cold exposures for 2 or 4 times significantly increased ME-sensitive hemagglutinin (IgM) production and markedly reduced ME-resistant (IgG) antibody levels (Figure 2). Four 30 minutes periods of cold initiated 12 hours after antigen injection significantly increased hemagglutinin levels and ME-sensitive
TOTAL ANTIBODY TITERS
ME-SENSTTIVE ANTIBODY TITERS
DAYS AFTER
SRBC
ME-RESISTANT ANYBODY TITER!
INJECTION
FIG. 4. Antibody titers of the birds exposed to four 30-minute periods of cold. ( - 1 2 refers cold treatments given 12 hours before the antigen injection; +12 refers cold treatments given 12 hours after the antigen injection).
The influence of temperature on antibody production has been investigated by several laboratories. The significant suppression of hemagglutinin production by chronic exposure to high temperatures during the pre-induction phase or at the time of antigen injection has been demonstrated (Thaxton et al., 1968; Subba Rao and Glick, 1970). These data complement the observation by Juszckiewicz (1967) where chickens infected with pasteurella and exposed to high temperature experience increased mortality associated with enhanced pasteurellosis and an emergence of secondary infections. On the other hand, chickens raised in a cold environment (2.2 C.) exhibited a lower infection of pasteurella. Now, we have demonstrated that chickens chronically ex-
TABLE 1. — Micrograms ofAbN/ml. of serum produced against bovine serum albumin (BSA) by chickens exposed to 30 minute periods of cold (7.2°C.) before (-12, -11, -10, -9) or after (+12, +13, +14, +15) antigen injection and chronic exposure at the time of antigen injection for 0—7 days' '2 Temperature
Precipitin resp.
Control - 1 2 , - 1 1 , - 1 0 , - 9 hours +12, +13, +14, +15 hours 0 - 7 days
104.40 119.73 181.29 169.34
1 2
± + ± +
30.12 b 14.53 b 21.21* 19.72a
Mean and standard deviation of 12 observations.
Means possessing different superscripts are significantly different at (P
Poultry Science 56:992-996, 1977 INTRODUCTION
Considerable research has been reported on changes in the physiology of domestic fowls exposed to stressful environmental temperatures. In all studies measurements of adrenal activity is frequently used as a criterion for physiological stress (Fortier, 1959; Fortier and DeGroot, 1959). The control of antibody-mediated immunity by varying environmental temperatures has been central to the research of our laboratory. Initial investigations in our laboratory identified the suppressive influence of acute heat on antibody production (Thaxton et al, 1968; Subba Rao and Glick, 1970). The natural exposure of birds to chronic periods of heat (or cold) occurs in many regions of the world. This prompted us to extend our immune studies to more prolonged periods of heat exposure. A search of the literature revealed a report of reduced infection to pasteurella in the presence of low temperature (Juszkiewicz, 1967). We included in our immune studies the exposure of birds to chronic and acute cold. MATERIALS AND METHODS Birds: Birds were from a commercial broiler flock or from a closed flock of New Hampshire, an American breed, maintained by Prof. L. J.
1
Please send all correspondence to: Bruce Glick, Ph.D., Professor, Poultry Science Department, P.O. Box 5188, Mississippi State University, Mississippi State, MS 39762.
Dreesen of our Poultry Department. Birds were reared either in all metal batteries placed in environmental chambers or on the floor of the environmental chambers with wood shavings as litter. The control birds were reared at ambient temperature. Environmental chambers: The environmental chambers were maintained by the South C e n t r a l Research Laboratory (U.S.D.A., A.R.S.). Each chamber (6.08 m. 2 floor area) was designed and built by Reece and Deaton (Reece and Deaton, 1967). The air exchange rate was 94.61/sec./chamber, which reduced ammonia, dust and carbon dioxide to a negligible factor, since ventilation was composed entirely of fresh air. The temperatures in the chambers were established at 7.2 C , 24 C , 32.2°C., 40.6°C. and 7.2°C.-32.2 C C. cycle over a 12 hour period with dewpoint temperature of 0°C. in 7.2°C. chamber and 10°C. in all other chambers. These constant conditions were maintained within ± 1 C. The light intensity for all treatments was an average of 1.2 foot candles at bird level for the first 3 weeks and 0.75 foot candles for the remaining time of the experimental period. Cold or Heat treatments: In the first set of experiments chickens were subjected to chronic cold or heat treatments from the day of hatch to 4 weeks of age in environmental chambers set at 7.2°C, 24°C, 7.2°C.-32.2°C. and 40.6 C. (Figure 1). In the second series of experiments the chickens were exposed to 30 minute periods of cold each hour for 2 or 4
992
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 31, 2015
ABSTRACT The effect of various environmental temperature changes on the antibody production of chickens was examined. Chronic cold exposure significantly increased the antibody titers. Birds exposed to 32.2°C. and above had significantly depressed agglutinin levels. Short term cold exposures 2 or 4 times following the antigen injection enhanced the agglutinin and hemolysin response. Thirty minute cold exposures for 2 or 4 times significantly increased IgM antibody production and markedly reduced the IgG antibody. The data suggest the elevation of antibody titers are in relation to time of the cold treatment and antigen injection. Observed differences of antibody production may be due to cold induced changes in the metabolic activity of antigen reactive cells and antibody producing cells.
993
COLD AND IMMUNE RESPONSE
— 7.2°c ••• 24°c — Control -»7.2°c-32.2°c — 32 2°c — 40.6°c
1 3
4
5
6
7
8
9
10
II
12
FIG. 1. Antibody titers of birds exposed to various environmental temperatures.
consecutive hours at the time of antigen injection (Figure 2). In the third series of experiments the birds were subjected to 30-minute periods of cold (Figure 4) either 12 hours before [-12, - 1 1 , - 1 0 , - 9 ] or 12 hours after [+12, +13, +15 hours] antigen administration. Antigens: The antigens used were sheep red blood cells (SRBC) and bovine serum albumin (BSA). SRBC stored in Alsever's solution were washed 3 times in physiological saline (0.9% NaCl) and resuspended to a 7 percent suspension. BSA was dissolved in saline to give a concentration of 40 mg./ml. Immunizations: All immunizations were made intravenously either with 1 ml. of the 7 percent suspension of SRBC or with BSA at the rate of 40 mg./kg. of body weight. In all experiments the type of antigen, the dosage of antigen, administration and vehicle, the time and methods of assessment of immune response were kept as constant as possible.
TOTAL ANTIBODY TITERS
ME-SENSITIVE ANTIBODY TITERS
DAYS AFTER
SRBC
ME-RESISTANT ANTIBODY TITERS
INJECTION
RESULTS FIG. 2. Antibody titers of birds exposed to short term cold treatments (chickens exposed to 30-minute periods of cold; CS-2, two times; CS-4, four times).
Chronic cold (7.2°C.) exposure significantly increased the antibody titers. Exposure to
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 31, 2015
2
DAYS AFTER SRBC INJECTION
Hemagglutinin assay: A microtiter procedure was employed for antibody titers (Wegman and Smithies, 1966). The birds were bled from the brachial vein 3 or 4 times between the 3rd and 10th day after immunization to collect serum for the micromethod. Serum samples were titrated individually (Subba Rao and Glick, 1975). Titers were expressed as the log 2 of the reciprocal of the highest dilutions giving visible agglutination. 2-Mercaptoethanol (ME) sensitive and resistant antibody: Equal volumes of serum and 0.2 m. ME in phosphate buffered saline (pH 7.4) were mixed and incubated at 37 C. for 30 minutes prior to serial dilution (Delhanty and Soloman, 1966). Agglutination tests were conducted and the titer was recorded as ME-resistant antibody. The reduction of the titer due to ME treatment was recorded as ME sensitive antibody. Hemolysin response: The hemolysin titers were determined using the microtiter procedure (Wegman and Smithies, 1966; Takatsy and Hamar, 1955). Serum dilutions were carried out in modified barbital buffer (Mayer et al., 1946). After the addition of SRBC, 25 jul. of complement was added to all the wells and the reaction mixture was incubated at 37 C. for 30 minutes. Titers were expressed as the log 2 like agglutinin levels. BSA precipitin levels: Birds were bled 7 days after the immunization. The antibody nitrogen (jug. AbN/ml.) in the serum was determined using a quantitative precepitin test (May and Glick, 1964). Plaque Forming Cell (PFC) assay: An agarless monolayer hemolytic plaque technique (Cunningham and Szenberg, 1968) as modified in our laboratory for the chicken system (Mueller et al., 1971) was employed. Four or five days following an I.V. injection of 1 ml. of a 7 percent suspension of SRBC, spleens were removed and antibody producing cells counted by the plaque forming assay (Subba Rao and Glick, 1975). The results were expressed as the number of PFC per 10 6 spleen cells. Statistical analysis: All the data were analyzed by analysis of variance (Steel and Torrie, 1960). Significant differences were determined by Duncan's new multiple range test (Duncan, 1955).
SUBBA RAO AND GLICK
994
DISCUSSION
DAYS AFTER SRBC INJECTION
FIG. 3. Hemolysin response of birds exposed to short term cold treatments (chickens exposed to 30-minute periods of cold; CS-2, two times; CS-4, four times). 24 C. increased the agglutinin levels but not significantly. Birds exposed to 32.2 C. and above experienced a significant suppression of their agglutinin levels (Figure 1). Birds exposed to cold 2 or 4 times immediately after the injection had significantly higher agglutinin and hemolysin responses than controls (Figures 2 and 3). The thirty minute cold exposures for 2 or 4 times significantly increased ME-sensitive hemagglutinin (IgM) production and markedly reduced ME-resistant (IgG) antibody levels (Figure 2). Four 30 minutes periods of cold initiated 12 hours after antigen injection significantly increased hemagglutinin levels and ME-sensitive
TOTAL ANTIBODY TITERS
ME-SENSTTIVE ANTIBODY TITERS
DAYS AFTER
SRBC
ME-RESISTANT ANYBODY TITER!
INJECTION
FIG. 4. Antibody titers of the birds exposed to four 30-minute periods of cold. ( - 1 2 refers cold treatments given 12 hours before the antigen injection; +12 refers cold treatments given 12 hours after the antigen injection).
The influence of temperature on antibody production has been investigated by several laboratories. The significant suppression of hemagglutinin production by chronic exposure to high temperatures during the pre-induction phase or at the time of antigen injection has been demonstrated (Thaxton et al., 1968; Subba Rao and Glick, 1970). These data complement the observation by Juszckiewicz (1967) where chickens infected with pasteurella and exposed to high temperature experience increased mortality associated with enhanced pasteurellosis and an emergence of secondary infections. On the other hand, chickens raised in a cold environment (2.2 C.) exhibited a lower infection of pasteurella. Now, we have demonstrated that chickens chronically ex-
TABLE 1. — Micrograms ofAbN/ml. of serum produced against bovine serum albumin (BSA) by chickens exposed to 30 minute periods of cold (7.2°C.) before (-12, -11, -10, -9) or after (+12, +13, +14, +15) antigen injection and chronic exposure at the time of antigen injection for 0—7 days' '2 Temperature
Precipitin resp.
Control - 1 2 , - 1 1 , - 1 0 , - 9 hours +12, +13, +14, +15 hours 0 - 7 days
104.40 119.73 181.29 169.34
1 2
± + ± +
30.12 b 14.53 b 21.21* 19.72a
Mean and standard deviation of 12 observations.
Means possessing different superscripts are significantly different at (P
