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Antibody-Mediated Immunity in the Presence of Mirex and DDT1,2
Antibody-Mediated Immunity in the Presence of Mirex and DDT 12 BRUCE G L I C K
Poultry Science Department, Mississippi Agricultural and Forestry Experiment Station, State University, Mississippi State, Mississippi 39762
Mississippi
(Received for publication November 15, 1973)
POULTRY SCIENCE 53: 1476-1485, 1974
E
NVIRONMENTAL exposure of vertebrates to 1,1, bis- (p-chlorophenyl) 2,2,2 -trichloroethane (p, p'DDT) and Mirex (dodecachlorooctahydro-l,3,3-metheno 2H cyclobuta (cd) pentaline) might take place by direct application of these pesticides or indirectly by breathing air or consuming food which contains the pesticides. While restrictions on the use of DDT are in force in the United States, its stability suggests that indirect exposure of vertebrates to DDT must be considered for many years (Woodwell, 1967). Mirex is an effective pesticide against the imported fire ant (Solenopsis saevissima richteri Forel). A program to eradicate the fire ant in the southeastern states by dispensing Mirex pellets from airplanes has been initiated (Markin et al., 1972). Thus vertebrates will be exposed to Mirex both directly
1. Supported, in part, by NSF grant GB 29220X. 2. Journal Article Number 2601 from the Mississippi Agricultural and Forestry Experiment Station.
and indirectly. Experiments designed to evaluate the immune system in the presence of Mirex have not been reported. On the other hand, fragmentary data are available on the immune system and tumor production for DDT and its analogues (Agthe et al, 1970; Fitzhugh and Nelson, 1947; Glick and Whatley, 1966; Kemeny and Tarjan, 1966; Tarjan and Kemeny, 1969; and Wasserman et al., 1969). In the chicken antibody-mediated immunity is dependent on the normal development of the bursa of Fabricius (Glick, 1970). Chemical interference with the bursa during embryonic development (Glick and Sadler, 1961; Mueller et al, 1960; and Warner et al, 1962) or neonatally (Glick, 1967, 1970, 1971; Lerman andWeidanz, 1970;andToivanen etal, 1972) will be reflected in an elimination or reduction in immunoglobulin G and antibody production and maturation of plasma cells. Therefore, the purpose of administering DDT or Mirex during the neonatal period (0-5 weeks of age) was to determine their potential for disrupting
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ABSTRACT The objective of these experiments was to evaluate the influence of Mirex (dodecachlorooctahydro-l,3,3,-metheno 2Hcyclobuta (CD) pentaline) and DDT (1,1, bis- (p-chlorophenyl) 2,2,2-trichloroethane) on antibody-mediated immunity in the chicken. Feeding 200-400 p.p.m. of Mirex for up to 5 weeks of age did not significantly influence body weight, weights of the bursa, spleen, adrenal, thymus, or liver, level of immunoglobulin M (IgM) or IgG, antibody response as measured by precipitin levels to bovine serum albumin (BSA) and number of plaque forming cells to sheep red blood cells or absolute number of lymphocytes and heterophils. Only body weight was significantly reduced by feeding 800 p.p.m. Mirex. Feeding 500 p.p.m. of Mirex significantly depressed the levels of IgG and IgM, but did not influence antibody production. DDT was lethal only at 1000 p.p.m. when 3/4 of all chicks died during the first 2 weeks after hatching. Feed withdrawal for 3-4 days before injecting BSA significantly depressed precipitin production and IgG levels in birds receiving 400 p.p.m. but not 200 p.p.m. of DDT. Feed withdrawal at the time of BSA injection and for the next 7 days did not influence birds receiving 200 p.p.m. DDT but significantly reduced titers in birds fed 400 p.p.m. DDT. The antibody response of Mirex birds (200 or 400 p.p.m.) was not modified. These data indicate that chickens exposed to high levels of DDT and faced with starvation and other possible environmental changes are less able to mount a normal antibody response.
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MATERIALS AND METHODS Pesticide Treatment and Analysis. Technical grade Mirex (M) and 99.0 + percent pure p, p' DDT (D) were obtained from Allied Chemical and Aldrich Chemical Companies, respectively. Weighed samples of each pesticide were added to a soybean oil premix and then combined with the basal feed (Morgan and Glick, 1972) to yield the desired levels e.g. 1 p.p.m. (M-l or D-l) and 400 p.p.m. (M-400 or D-400). In one trial Mirex was dissolved in acetone, added to the basal feed and the acetone evaporated. Liver samples were analysed for p,p'-DDE; p,p'-DDD; o,p'-DDT; p,p'-DDT; and Mirex (Thompson, 1971). The chickens were from a strain of New Hampshire, an American breed, developed by Professor Dreesen of our Poultry Department. All eggs were hatched in James way incubators. Histological Data. The bursa of Fabricius and thymus were stained with hematoxylin and eosin while the spleen and gland of Harder were fixed in Carnoy's and stained with methyl green pyronin (Opstad, 1959). The latter stain reveals the presence of plasma cells or the antibody producing cells. AH glands, adrenal, bursa, gland of Harder, spleen, liver and thymus, were weighed to the nearest 0.1 mg. and converted to mg. /gm. of body weight for the analysis of variance.
Significant mean differences were revealed by Duncan's new multiple range test (1955). Immunological and Hematological Data. Total white blood cell counts were made by the method of Natt and Herrick (1958) and differential counts, percentage of lymphocytes and heterophils, determined by counting 100 cells from a blood-smear stained with Wright's stain. The capillary pipette method was employed to determine hematocrit values (McGovern et al., 1955). Birds were immunized IV with 40 mg. of bovine serum albumin (BSA) per 100 gm. of body weight and bled 7 days after the injection. The quantitative precipitin test revealed the mg. antibody nitrogen per ml. (ng. AbN/ml.) of serum in the birds receiving BSA (May and Glick, 1964). The monolayer technique of Cunningham and Szenberg (1968) has been modified in our laboratory (Sato and Glick, 1970; and Mueller et al., 1971) and used routinely for the demonstration of antibody producing cells. Four days following an I.V. injection of 1 ml. of a 7% suspension of sheep-red-blood cells (SRBC) spleens were removed and antibody producing cells counted by the plaque forming assay. The phagocytic ability of DDT treated birds was assessed by administering 16 mg. of carbon (Pelikan CI 1/1431a, Gunther Wagner) per 100 gm. of body weight (Subba Rao and Glick, 1970). Significant treatment differences for all parameters were determined by Duncan's new multiple range test (1955). The levels of immunoglobulin G (IgG and M (IgM) were determined by disc electrophoresis (Glick, 1968; and Morgan and Glick, 1972). RESULTS Mirex. Feeding Mirex from hatch to 7 days of age at 1, 20 or 200 p.p.m. did not influence hematocrit levels, feed consumption or body weights at 1 week of age or weights of body, bursa, spleen, and comb two weeks after
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bursal development and future antibody production. In the present work histological data, growth, and immunological parameters in chicks treated neonatally with varying levels of DDT and Mirex have been studied. The chicks revealed a greater sensitivity to DDT than to Mirex. However, the pesticide levels and conditions necessary to produce deleterious effects on the histology and the immune system were extremely high and severe. A portion of this work has been reported (Glick, 1972b).
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TABLE 1.—The response of chicks fed Mirex (M) from hatch to 1 week of age P.P.M. of Mirex Body wt, gm. 1 wk. 3 wks. 5 wks. Feed consumed 0-7 days; lb.
M-l
M-20
M-200
104.4 (30) 361.1 (25) 773.9 (10)
105.5 (32) 368.3 (26) 741.4(14)
107.3 (29) 382.6 (23) 810.4 (12)
107.1 (30) 390.3 (24) 827.4 (12)
4.75
5.00
5.50
4.50
3.41 ±0.53 1.08±0.35 113.3 26.2±1.5
2.51+0.39 1.09±0.32 123.8 29.9+3.6
3.14±0.78 0.97±0.24 113.2 26.8+2.6
3.18+0.62 0.97±0.27 129.1 28.5±3.9
0 0
0.225; 0.215 .004; Trace
4.191; 4.155 .069; .060
47.027; 47.102 .582; .464
P.P.M. in liver 1 wk. of age 5 wks. of age a
( ) = number of birds. No significant mean differences. All means accompanied by a standard deviation. a - 4 weeks after Mirex withdrawal from feed. Mirex withdrawal (Table 1). Liver residues of Mirex were in the same ratio as feed levels and were markedly reduced 4 weeks after Mirex withdrawal from the feed. Increasing the level of Mirex to 400 and 800 p.p.m. and extending the feeding period over the first 5 weeks of life did not compromise the weights of the adrenal, bursa, liver, thymus
or spleen (Tables 2 and 3). The M-800 treatment significantly reduced body weight (Table 3). Antibody-mediated immunity as evaluated by production of precipitin to BSA, levels of IgG and IgM, and number of antibody producing cells (plaque forming cells, PFC) in response to SRBC was not affected by the Mirex treatment (Tables 2 and 3). Liver
TABLE 2.—Body and gland weights and antibody response of chickens receiving 400 p.p.m. of Mirex (M) from hatch to 5 weeks of age P.P.M. of Mirex M-0
M-400
Body wt. 2 wk. 4 wk.
201.4 (22) 513.2 (15)
188.5 (25) 527.2 (18)
2 weeks (6) Bursa mg./gm. Thymus mg./gm. Spleen mg./gm. Liver mg./gm.
2.87±0.75 0.43+0.11 1.70±0.73 3.33±0.25
3.01 ±0.57 0.40±0.11 1.22±0.30 3.12+0.16
1.17+0.22 3.46±0.83 20.58±3.50 80.78±43.10 603,903,2586 .026; .028; .023; .024
1.21±0.35 3.52±0.82 21.65±3.61 95.90±44.20 1277,1567,2300 19.07; 21.30; 63.15 27.14; 30.14; 23.4
5 weeks (8) Bursa Spleen Liver ixg. AbN/ml. (7) P F C / 10s spleen cells PPM in liver No significant differences. ( ) = number of birds.
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3 wks. of age (6) Bursa, mg./gm. Spleen mg./gm. Comb, mg. Hematocrit-1 wk.
M-0
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TABLE 3.—Body and gland weights and serum protein analysis of 5-week-old Mirex fed birds P.P.M. ( )f Mirex M-400 M-200 607b 633" 0.92±0.34 1.98+0.80 0.51±0.11 0.51+0.11 4.51±1.63 3.49±1.10
M-800 537c 647a 0.84±0.22 0.98+0.31 0.65+0.22 0.57±0.08 2.87±0.96 3.00+0.91 3.75±0.58 3.77±0.35 1.46±0.30 1.39+0.20 676±352 817.20±257 562 ±234 428.0±90 403±141 258.0±122 NM <100 >90 NM .075 Means possessing different superscripts are significantly different at the 5% level. Eight birds per mean. M-0
Body wt., gm. Bursa, mg./gm. Adrenal mg./gm. Spleen mg./gm. Total protein gm.% Albumin gm.% IgG mg.% IgM mg.% Transferrin mg.% P.P.M. in liver
T A B L E 4.-
FIG. 2. Bursal follicles from a control chick exhibiting normal basophilic cells and division of bursal follicles into medulla and cortex (x 200).
- White blood cell counts of Mirex (M) fed birds before and after the injection of four I. U. ACTH per 100 gm. body weight
TWBC x HF 2 wks. 3 wks. 4 wks.*
M-0
P.P.M. Mirex M-200
M-400
17.8 16.4 23.8
14.6 22.0 21.7
12.9 17.9 20.5
Heterophil x /0 3 4.4 5.8 2 wks. 4.9 4.9 3 wks. 12.0 4 wks.* 14.3 TWBC—Total-white-blood cell count. * = 4 I.U. ACTH/100 gm. body weight. Injected 4 hours before cell counts. No significant treatment or treatment x week differences.
2.0 4.4 12.3
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FIG. 1. Bursal follicles from a bird 3 weeks old treated with 400 p.p.m. of Mirex exhibiting pale lymphoid cells similar to cyclophosphamide-treated chicks (x 200).
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TABLE 5.—Body, gland, serum protein, and antibody response of chicks receiving 500 p.p.m. of DDT (D) hatch to 5-weeks of age P.P.M. D-0 Body wt., gm. 1 wk.
2 wks. 4 wks. 2 wks. of age (12) Bursa mg. /gm. Adrenal mg./gm. Spleen mg./gm. Liver mg./gm. Comb mg.
86.78±12.14(37) 240.55±57.1 (27) 513.57±88.9 (14)
85.36±4.23 (30) 241.44±26.3 (27) 508.93±77.0(15)
2.77±0.65 0.15±0.03 1.23+0.41 33.12+12.90 79.36*±19.4
2.93±0.55 0.15+0.05 1.25+0.41 35.80* ±1.96 54.56±17.6
86.76±34.60 1883,992,1897 11.47 764.1*±55.2 311.0* ±74.1 226.54±82.2
86.65±30.6 75.81, 1106,2643 16.12 403.40±246.0
187.8± 142.0 145.44±67.7
slight reduction in the number of lymphoid cells found in the medulla of bursal follicles. By 3 weeks of age the bursal follicles of M-400 chicks revealed the presence of pale or lightly staining lymphoid cells, some dissolution of lymphoid cells and indipping of
residues of Mirex in the M-400 group varied in one experiment from 19.07-63.15 p.p.m. (Table 2) while they remained above 90 p.p.m. and below 100 p.p.m. in the other experiment (Table 3). Histologically the bursa from 2 week old M-400 treated chicks exhibited a
TABLE 6.—Body weight, gland weight, and antibody response of birds fed varying levels of DDT (D) from hatch to six weeks of age P.P.M D-400
D-800
0-1600°
98.21±58.51
75.68±24.90
108.09±53.01
81.29± 19.20
941.42 0.53±0.20 32.34±3.10 0.085±00.010 2.01 ±0.54 1.87
903.00 0.46±0.07 28.06±2.10 0.090±0.030 1.73 ±0.37 1.80
902.57 0.53±0.14 32.58+2.93 .097±0.087 2.07±0.66 1.75
D-0 5 weeks of age, 8 birds/ mean M-g. AbN/ml. 6 weeks of age, 7 birds/ mean Body, gm. Bursa, mg./gm. Liver, mg./gm. Adrenal, mg./gm. Spleen, mg./gm. Feed efficiency p.p.m. in liver P.p-DDE p,p'-DDD p,p'-DDT Total DDT
<0.10
7.1 10.2 7.0 24.2
12.0 12.0 4.3 28.3
38.9 46.6 34.7 44.5 8.3 5.6 82.0 96.7
21.7 44.9 15.2 81.8
22.5 19.0 36.5 44.3 21.0 4.6 80.0 68.3
a—Data from birds receiving 1600 p.p.m. from 1-6 weeks of age. This level lethal to chicks when initiated at hatching, see text.
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4 wks. of age M-g. AbN/ml. (8) PFC/106 spleen cells Phagocytic index (6) IgG mg.% (8) IgM mg.% (8) Transferrin mg.% (8) () = number of birds. *P < .05.
D-500
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epithelial cells lining the bursal folds (Figures 1 and 2). The methyl green-pyronin (MGP) stain revealed the presence of plasma cells in the spleen, caecal tonsil, and gland of Harder. The total white blood cell count and absolute number of heterophils in 2 and 3-week-old M-200 and M-400 treated chicks were similar to control values (Table 4). The three groups exhibited the normal increase in absolute number of heterophils upon challenge with 4 I.U. ACTH/100 gm. of body weight (Table 4). FIG. 3. Bursal follicles from a 3-week-old bird treated with 500 p.p.m. of DDT exhibiting dissolution of bursal follicles, vacuolation and loss of medullary cells (x 200). remaining two died in the fourth week with liver residues of 1084.8 (p,p'DDE-685.1 p.p.m. and p,p'DDD-397.6 p.p.m.) and 526.7 (p,p'DDE-301.7 p.p.m. and p,p'DDD-221.7 p.p.m.) total p.p.m. of DDT. All eight chickens exhibited ataxia within one week of death. At 1 week of age 1600 p.p.m. of DDT was added to the basal diet of 7 chicks. Within two weeks, 4 of these chicks exhibited ataxia. At 5 weeks of age, 4 weeks after receiving 1600 p.p.m. of DDT, these chickens con-
TABLE 7.—Precipitin response to BSA ("/xg. AbN/ml.) of 5-week-old DDT (D)-treated chickens (6 per mean) Feed not withheld Groups
D-0 p.p.m. D-200 p.p.m. D-400 p.p.m.
111.35b±34.90
Feed withheld during Induction and proPreinduction duction period period (4d. before (At time of BSA BSA inj.) Inj. and for next 7 days) 67.0C±44.40 (0.04±0.02) 33.03de±23.01 15.13e±8.10
Means possessing different superscripts are significantly different (p < .01). ( ) = p.p.m. of total DDT in liver from separate birds at 6 weeks of age.
118.40a±62.10 115.44b±74.50 (32.26± 14.35) 40.16d±2.00 (40.12±28.73)
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DDT. DDT, like Mirex, when administered at levels up to 800 p.p.m. failed to affect the weights of the adrenal, bursa, thymus, or spleen (Tables 5 and 6). Comb size (equal numbers of males and females) was significantly decreased (Table 5) and liver size significantly increased in one of two experiments (Tables 5 and 6). Unlike Mirex-fed birds, the DDT treated chickens exhibited significantly depressed levels of IgG and IgM (Table 5). However, the DDT-treated birds ability to produce antibody to BSA and PFC to SRBC was not significantly different from control animals (Tables 5 and 6). Administering 1600 p.p.m. of DDT at hatching was lethal to 6 of 8 chicks by two weeks of age. The
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TABLE 8.—Immunoglobulin G and IgM levels (mg.%) of 6-week-old birds fed DDT (D) for 5 weeks and starved from week 5 to week 6 Feed withheld
Immunoglobulin IgG
Feed not withheld(D-O) 720.30±81.10
D-0 7O6.90±21.70
D-200 699.16±65.30
D-400 527.32±97.70
IgM
251.8±47.70
196.06±68.60
256.8±8.00
293.00± 187.30
Means not underlined by the same line are significantly different at the 1% level.
Feed Withdrawal and Pesticides. Withdrawing feed from the D-200 and D-400 birds four days before the administration of BSA (preinduction period) significantly depressed the titers below comparable controls and controls on full feed (Table 7). Also, the controls (D-0) experienced a significant reduction in antibody titers when feed was withdrawn during the preinduction period. The concentration of IgG (mg.%) was signif i-
TABLE 9.—Precipitin response (fxg. AbN/ml.) of 5-week-old Mirex fed chickens (7 per mean)
Groups M-Op.p.m.
Feed not withheld 123.3±52.50 (0.13±0.14)
M-200p.p.m.
96.18±59.40 128.85±36.50 (75.59±76.55) 122.84±77.80 (250.93+405.88)
M-400 p.p.m. M-"200" p.p.m.
Feed withheld 3 days prior to BSA injection (preinduction period)
116.35+87.8
M-"200"—Mirex added to feed in acetone. No significant differences observed. ( ) = p.p.m. Mirex in liver from two separate birds at 6 weeks of age.
cantly depressed in the D-400 group (Table 8). Withdrawing feed at the time of BSA injection and for a seven day period (induction and production periods) significantly enhanced titers in the D-0 group, did not influence titers in the D-200 birds, and significantly depressed titers in the D-400 group (Table 7). On the other hand, withdrawing feed from the M-0, M-200 and M-400 birds three days before the administration of BSA (preinduction period) did not significantly influence the preciptin levels (Table 9). Body weights were significantly depressed during starvation in all groups. For example, body weight before feed withdrawal and seven days later was 840.71 ± 61.60 and 601.85 ± 42.00 for D-0; 776.80 ± 81.61 and 547.62 ± 49.22 for D-200; and 787.50 ± 96.61 and 557.37 ± 58.20 for D-400. No mortality was experienced. Also, withdrawing feed for three days from the D-0, D-200, D-400 groups did not significantly increase the number of absolute heterophils over full fed birds. DISCUSSION We have demonstrated that the bursa is capable of regeneration following steroid induced regression during the neonatal period (Glick, 1967, 1970). The regeneration of the bursa was delayed as evidenced by a marked suppression of hemagglutinin and precipitin production at 5 weeks of age and a normal titer of these antibodies by 8 weeks of age. While steroid administration to neonatal chicks eliminated or markedly reduced the
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tained DDT-liver residues comparable to chicks fed 800 p.p.m. from hatching (Table 6). The D-500 chicks exhibited severe dissolution of bursal follicles by 3 weeks of age with vacuolation and loss of medullary cells (Figure 3). However, a second experiment revealed no obvious difference in D-500 and D-0 bursae at 3 weeks of age. The MGP stain revealed the presence of plasma cells in both the spleen and caecal tonsils.
ANTIBODY-MEDIATED IMMUNITY
Failure of excessive levels of DDT or Mirex to markedly suppress antibody-mediated immunity demonstrates that in the chicken, whose immune system has been used as a model to understand the mammalian immune system, acute exposure to DDT or Mirex will not significantly compromise humoral immunity. Our data did not include functional studies of cell-mediated immunity, but the normal histology of the thymus which controls cell-mediated immunity (Cooper et al., 1966) from DDT and Mirex birds suggests an intact cell-mediated system in the pesticide-treated birds. DDT-treated birds but not Mirex birds appeared to be sensitive to feed withdrawal. Starvation during the preinduction period significantly depressed antibody response in DDT-treated birds (D-200 and D-400) over comparable controls (D-0) and full-fed controls. Also, in this experiment the controls (D-0) starved during the preinduction period experienced a significant reduction in antibody titers when compared to full-fed controls. These data indicate that chickens exposed to high levels of DDT and faced with starvation and other possible environmental changes are less able to mount a normal antibody response. While Donaldson et al. (1968) reported mortality of DDT-fed birds after four days of starvation none of our DDT or Mirex-treated birds died during the 4-7 day starvation periods. Also, DDT and Mirex treatment did not produce physiological stress as evidenced by a lack of change in the absolute number of heterophils. There remain two avenues of study with DDT and Mirex. The first entails chronic treatment of birds with low levels of the pesticides and the assessment of the immune system of the adult and their offspring. The former has been initiated. The second approach is to administer the two pesticides to the embryo by in ovo injections or shell application and then to study the fine structure of the bursa and thymus in the embryo
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presence of bursal follicles (Glick, 1967, 1970), the alkylating agent, cyclophosphamide, did not destroy the cytoarchitecture of the bursa (Glick, 1971; Linna et al., 1972). However, cyclophosphamide administered during the first 2-3 days of the chicks life will produce agammaglobulinemia (Lerman and Weidanz, 1970; Glick, 1971; and Toivanen et al., 1972). Therefore, marked interference with bursal development during the first week of life will be reflected in varying degrees of humoral immunity suppression. Hence, the purpose of administering Mirex and DDT to neonatal chicks was to assess their potential for disrupting bursal development and future humoral immunity. Since these pesticides proved unsuccessful in initiating bursal involution during the first 2 weeks of life, one might conclude that the pesticides would not alter immunoglobulin (Ig) or antibody production. And, while this was true of Mirex it was not true for DDT. The latter significantly reduced IgG and IgM. Glick and Whatley (1966) demonstrated that o,p'DDD would suppress the precipitin response in chickens. However, the significance of this report is questionable since the DDT-analogue was administered intravenously. In rabbits receiving 200 p.p.m. of DDT in the drinking water, a significant reduction in gamma globulin was noted (Wasserman et al., 1969). These authors report individual titer values for the rabbits injected with ovalbumin and while the titers were lower in the DDT-treated rabbits by one tube my analysis of the raw data demonstrated no statistical significance. Degeneration of bursal follicles by three weeks cannot explain the Ig results in DDT-treated birds since the same regression occurred in Mirex birds without the accompanying reduction in Ig levels. The possibility that the spleen or other sites may be sensitive to increased loads of organic chemicals during the neonatal period (Glick, 1972a) might explain the DDT effect in the absence of an obvious bursal response.
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as well as antibody and cell-mediated immunity in the hatched chick. ACKNOWLEDGEMENT
REFERENCES Agthe, C., H. Garcia, P. Shubik, L. Tomatis and E. Weyon, 1970. Study of the potential carcenogenicity of DDT in the Syrian golden hamster. Proc. Soc. Exptl. Biol. Med. 134: 113-116. Cooper, M. D., R. D. A. Peterson, M. A. South and R. A. Good, 1966. The functions of the thymus system and bursa system in the chicken. J. Exp. Med. 123: 75-102. Cunningham, A. J., and A. Szenberg, 1968. Further improvements in the plaque technique for detecting single antibody producing cells. Immunology, 14: 599-600. Donaldson, W. E., T. J. Sheets and M. D. Jackson, 1968. Starvation effects on DDT residues in chick tissues. Poultry Sci. 47: 237-243. Duncan, D. B., 1955. Multiple range and multiple " F " tests. Biometrics, 11: 1-42. Fitzhugh, O. G. and A. A. Nelson, 1947. The chronic oral toxicity of DDT (2,2, bis (p-chlorophenyl-1,1,1 trichloroethane). J. Pharmac. Exp. Ther. 89: 18. Glick, B., 1967. Antibody and gland studies in cortisone and ACTH-injected birds. J. Immun. 98: 10761084. Glick, B., 1968. Serum protein electrophoresis patterns in acrylamide gel: patterns from normal and bursaless birds. Poultry Sci. 47: 807-814. Glick, B., 1970. Immunity studies in testosterone propionate injected chicks. Int. Arch. Allergy, 38: 93-103. Glick, B., 1970. The bursa of Fabricius: A central issue. BioScience, 20: 602-604. Glick, B., 1971. Morphological changes and humoral immunity in cyclophosphamide-treated chicks.
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I thank Mr. F. L. Bailey of the Allied Chemical Corporation and the Aldrich Chemical Company for generously supplying us with Mirex and DDT, respectively. The technical assistance of Sharon Brock, Diana Weigmann, and Sandra Whatley contributed to the success of this project. Special acknowledgement is made to the department of Biochemistry, Mississippi State University, Dr. Ben Barrentine, Head, and Research Chemist Jimmie Cain for the liver residue analyses.
Transplantation, 11: 433-439. Glick, B., 1972a. Cortisone, age and antibody-mediated immunity. Int. Arch. Allergy, 43: 766-771. Glick, B., 1972b. The immunobiological influence of Mirex and DDT. Poultry Sci. 51: 1861. Glick, B., and C. R. Sadler, 1961. The elimination of the bursa of Fabricius and reduction of antibody production in birds from eggs dipped in hormone solutions. Poultry Sci. 40: 185-189. Glick, B., and S. Whatley, 1966. The effect of o,p-DDD in the chicken. Experientia, 2: 179-182. Kemeny, T., and R. Tarjan, 1966. Investigations on the effects of chronically administered small amounts of DDT in mice. Experientia, 22: 748. Lerman, S. P., and W. Weidanz, 1970. The effect of cyclophosphamide on the ontogeny of the humoral immune response in chickens. J. Immunol. 105: 614-619. Linna, T. J., D. Frommel and R. A. Good, 1972. Effect of early cyclophosphamide treatment on the development of lymphoid organs and immunological functions in the chicken. Int. Arch. Allergy, 42: 20-39. Markin, G. P., J. H. Ford, J. C. Hawthorne, J. H. Spence, J. Davis and H. L. Collins, 1972. Insecticide Mirex and techniques for its monitoring. U.S.D.A. Bull. APHIS 81 3 pp. 19. May, D., and B. Glick, 1964. Weight of the bursa of Fabricius and antibody response of chicks hatched from eggs dipped in varying concentrations of testosterone propionate. Poultry Sci. 43:450-453. McGovern, J. J., A. R. Jones and A. G. Steinberg, 1955. The hematocrit of capillary blood. New England J. Med. 253: 308-312. Morgan, G. W., Jr., andB. Glick, 1972. A quantitative study of serum proteins in bursectomized and irradiated chickens. Poultry Sci. 51: 771-778. Mueller, A. P., K. Sato and B. Glick, 1971. The chicken lacrimal gland, gland of Harder, caecal tonsil, and accessory spleens as sources of antibody-producing cells. Cellular Immunology, 2: 140-152. Mueller, A. P., H. P. Wolfe and R. K. Meyer, 1960. Precipitin production in chickens. XXI. Antibody production in bursectomized chickens and in chickens injected with 19-nortestosterone on the fifth day of incubation. J. Immunol. 85: 172-179. Natt, M. P., and C. A. Herrick, 1958. A new blood diluent for counting the erythrocytes and leukocytes of the chicken. Poultry Sci. 31: 735-738. Opstad, A. M., 1959. A methyl green-pyronin stain for plasma cells in tissues. Stain Technol. 34: 293. Sato, K., and B. Glick, 1970. Are splenic plaque forming cells sensitive to heat in vitro? Life Sci. 9: 175-180. Subba Rao, D. S. V., and B. Glick, 1970. Immunosup-
ANTIBODY-MEDIATED IMMUNITY
pressive action of heat in chickens. Proc. Soc. Exptl. Biol. Med., 133: 445-448. Tarjan, R., and T. Kemeny, 1969. Multigeneration studies of DDT in mice. Fd. Cosmet. Toxicol. 7: 215-222. Thompson, J. F., 1971. Micro methods for chlorinated hydrocarbon pesticides and metabolites for human tissue and excreta, section 5A (2A). In: Manual of Analytical Methods. Published by Perrine Laboratories, Florida. Toivanen, P., A. Toivanen, T. J. Linna and R. A. Good, 1972. Ontogeny of bursal function in chick-
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ens. II. Post embryonic stem cell for humoral immunity. J. Immunol. 109: 1071-1080. Warner, N. L., A. Szenberg and F. M. Burnet, 1962. The immunological role of different lymphoid organs in the chicken. I. Dissociation of immunological responsiveness. Aust. J. Exp. Biol. 40: 373-388. Wasserman, M., D. Wasserman, Z. Gershon and L. Zellermayer, 1969. Effects of organochlorine insecticides on body defense systems. Ann. New York Acad. Sci. 160: 393-401. Woodwell, G. B., 1967. Toxic substances and ecological cycles. Sci. Amer. 216: 24-36.
D . C. BORRON, M . G. MCCARTNEY AND H . L . FULLER
Department of Poultry Science, University of Georgia, Athens, Georgia 30602 (Received for publication November 15, 1973)
ABSTRACT These studies were undertaken to determine the effects of restricted energy feeding during the growing period upon the subsequent reproductive performance of Large White turkey females. Energy intake was limited to two-thirds of that voluntarily consumed by birds receiving practical-type growing diets ad libitum. Dietary treatments were imposed from either 8 to 32, 12 to 24, or 12 to 28 weeks of age under either natural increasing, natural decreasing, or constant eight-hour daylength. Data were collected on the number of days after the onset of stimulatory light to first egg and to 50 percent egg production, and on body weight at the end of the restriction period, at 50 percent egg production, and at the end of the reproductive period. Data were also collected on shank length, egg production, egg weight, fertility, hatchability, number of poults per hen, and carcass composition at the end of the restriction period and at 50 percent egg production. Females grown on restricted energy diets were less fat, had shorter shanks and weighed less at the end of the restriction period. Carcass fat, however, was not significantly affected at the time of 50 percent egg production. Only a slight delay in sexual maturity was noted in those birds grown on restricted energy diets. Restricting energy during the growing period did not affect reproductive performance. POULTRY SCIENCE 53: 1485-1493, 1974
T
URKEY poults grown for breeding stock are normally provided the same diets fed market birds until just prior to the time they are brought into egg production. Growing and finishing diets are designed to give maximum growth and finish at market age and, consequently, may not be optimum for growing turkeys that will be used for breeders for the production of hatching eggs. Several feeding programs are being used by producers of hatching eggs to delay sexual maturity and to improve the reproductive
performance of the chicken. Quantitative feed restriction during the rearing period has been shown to delay sexual maturity and improve reproductive performance of both broiler breeder and egg-type chickens (Isaacks et al., 1960; Hollands and Gowe, 1961; Fuller and Dunahoo, 1962; and Strain et al., 1965). Low protein diets have also been found to be effective in delaying maturity and improving reproductive performance of the chicken (Wright et al., 1968). Dietary energy restriction also has been shown to
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The Effects of Restricted Energy Feeding During the Growing Period on the Reproductive Performance of Turkey Breeder Females
Poultry Science Department, Mississippi Agricultural and Forestry Experiment Station, State University, Mississippi State, Mississippi 39762
Mississippi
(Received for publication November 15, 1973)
POULTRY SCIENCE 53: 1476-1485, 1974
E
NVIRONMENTAL exposure of vertebrates to 1,1, bis- (p-chlorophenyl) 2,2,2 -trichloroethane (p, p'DDT) and Mirex (dodecachlorooctahydro-l,3,3-metheno 2H cyclobuta (cd) pentaline) might take place by direct application of these pesticides or indirectly by breathing air or consuming food which contains the pesticides. While restrictions on the use of DDT are in force in the United States, its stability suggests that indirect exposure of vertebrates to DDT must be considered for many years (Woodwell, 1967). Mirex is an effective pesticide against the imported fire ant (Solenopsis saevissima richteri Forel). A program to eradicate the fire ant in the southeastern states by dispensing Mirex pellets from airplanes has been initiated (Markin et al., 1972). Thus vertebrates will be exposed to Mirex both directly
1. Supported, in part, by NSF grant GB 29220X. 2. Journal Article Number 2601 from the Mississippi Agricultural and Forestry Experiment Station.
and indirectly. Experiments designed to evaluate the immune system in the presence of Mirex have not been reported. On the other hand, fragmentary data are available on the immune system and tumor production for DDT and its analogues (Agthe et al, 1970; Fitzhugh and Nelson, 1947; Glick and Whatley, 1966; Kemeny and Tarjan, 1966; Tarjan and Kemeny, 1969; and Wasserman et al., 1969). In the chicken antibody-mediated immunity is dependent on the normal development of the bursa of Fabricius (Glick, 1970). Chemical interference with the bursa during embryonic development (Glick and Sadler, 1961; Mueller et al, 1960; and Warner et al, 1962) or neonatally (Glick, 1967, 1970, 1971; Lerman andWeidanz, 1970;andToivanen etal, 1972) will be reflected in an elimination or reduction in immunoglobulin G and antibody production and maturation of plasma cells. Therefore, the purpose of administering DDT or Mirex during the neonatal period (0-5 weeks of age) was to determine their potential for disrupting
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ABSTRACT The objective of these experiments was to evaluate the influence of Mirex (dodecachlorooctahydro-l,3,3,-metheno 2Hcyclobuta (CD) pentaline) and DDT (1,1, bis- (p-chlorophenyl) 2,2,2-trichloroethane) on antibody-mediated immunity in the chicken. Feeding 200-400 p.p.m. of Mirex for up to 5 weeks of age did not significantly influence body weight, weights of the bursa, spleen, adrenal, thymus, or liver, level of immunoglobulin M (IgM) or IgG, antibody response as measured by precipitin levels to bovine serum albumin (BSA) and number of plaque forming cells to sheep red blood cells or absolute number of lymphocytes and heterophils. Only body weight was significantly reduced by feeding 800 p.p.m. Mirex. Feeding 500 p.p.m. of Mirex significantly depressed the levels of IgG and IgM, but did not influence antibody production. DDT was lethal only at 1000 p.p.m. when 3/4 of all chicks died during the first 2 weeks after hatching. Feed withdrawal for 3-4 days before injecting BSA significantly depressed precipitin production and IgG levels in birds receiving 400 p.p.m. but not 200 p.p.m. of DDT. Feed withdrawal at the time of BSA injection and for the next 7 days did not influence birds receiving 200 p.p.m. DDT but significantly reduced titers in birds fed 400 p.p.m. DDT. The antibody response of Mirex birds (200 or 400 p.p.m.) was not modified. These data indicate that chickens exposed to high levels of DDT and faced with starvation and other possible environmental changes are less able to mount a normal antibody response.
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ANTIBODY-MEDIATED IMMUNITY
MATERIALS AND METHODS Pesticide Treatment and Analysis. Technical grade Mirex (M) and 99.0 + percent pure p, p' DDT (D) were obtained from Allied Chemical and Aldrich Chemical Companies, respectively. Weighed samples of each pesticide were added to a soybean oil premix and then combined with the basal feed (Morgan and Glick, 1972) to yield the desired levels e.g. 1 p.p.m. (M-l or D-l) and 400 p.p.m. (M-400 or D-400). In one trial Mirex was dissolved in acetone, added to the basal feed and the acetone evaporated. Liver samples were analysed for p,p'-DDE; p,p'-DDD; o,p'-DDT; p,p'-DDT; and Mirex (Thompson, 1971). The chickens were from a strain of New Hampshire, an American breed, developed by Professor Dreesen of our Poultry Department. All eggs were hatched in James way incubators. Histological Data. The bursa of Fabricius and thymus were stained with hematoxylin and eosin while the spleen and gland of Harder were fixed in Carnoy's and stained with methyl green pyronin (Opstad, 1959). The latter stain reveals the presence of plasma cells or the antibody producing cells. AH glands, adrenal, bursa, gland of Harder, spleen, liver and thymus, were weighed to the nearest 0.1 mg. and converted to mg. /gm. of body weight for the analysis of variance.
Significant mean differences were revealed by Duncan's new multiple range test (1955). Immunological and Hematological Data. Total white blood cell counts were made by the method of Natt and Herrick (1958) and differential counts, percentage of lymphocytes and heterophils, determined by counting 100 cells from a blood-smear stained with Wright's stain. The capillary pipette method was employed to determine hematocrit values (McGovern et al., 1955). Birds were immunized IV with 40 mg. of bovine serum albumin (BSA) per 100 gm. of body weight and bled 7 days after the injection. The quantitative precipitin test revealed the mg. antibody nitrogen per ml. (ng. AbN/ml.) of serum in the birds receiving BSA (May and Glick, 1964). The monolayer technique of Cunningham and Szenberg (1968) has been modified in our laboratory (Sato and Glick, 1970; and Mueller et al., 1971) and used routinely for the demonstration of antibody producing cells. Four days following an I.V. injection of 1 ml. of a 7% suspension of sheep-red-blood cells (SRBC) spleens were removed and antibody producing cells counted by the plaque forming assay. The phagocytic ability of DDT treated birds was assessed by administering 16 mg. of carbon (Pelikan CI 1/1431a, Gunther Wagner) per 100 gm. of body weight (Subba Rao and Glick, 1970). Significant treatment differences for all parameters were determined by Duncan's new multiple range test (1955). The levels of immunoglobulin G (IgG and M (IgM) were determined by disc electrophoresis (Glick, 1968; and Morgan and Glick, 1972). RESULTS Mirex. Feeding Mirex from hatch to 7 days of age at 1, 20 or 200 p.p.m. did not influence hematocrit levels, feed consumption or body weights at 1 week of age or weights of body, bursa, spleen, and comb two weeks after
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bursal development and future antibody production. In the present work histological data, growth, and immunological parameters in chicks treated neonatally with varying levels of DDT and Mirex have been studied. The chicks revealed a greater sensitivity to DDT than to Mirex. However, the pesticide levels and conditions necessary to produce deleterious effects on the histology and the immune system were extremely high and severe. A portion of this work has been reported (Glick, 1972b).
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B.GLICK
TABLE 1.—The response of chicks fed Mirex (M) from hatch to 1 week of age P.P.M. of Mirex Body wt, gm. 1 wk. 3 wks. 5 wks. Feed consumed 0-7 days; lb.
M-l
M-20
M-200
104.4 (30) 361.1 (25) 773.9 (10)
105.5 (32) 368.3 (26) 741.4(14)
107.3 (29) 382.6 (23) 810.4 (12)
107.1 (30) 390.3 (24) 827.4 (12)
4.75
5.00
5.50
4.50
3.41 ±0.53 1.08±0.35 113.3 26.2±1.5
2.51+0.39 1.09±0.32 123.8 29.9+3.6
3.14±0.78 0.97±0.24 113.2 26.8+2.6
3.18+0.62 0.97±0.27 129.1 28.5±3.9
0 0
0.225; 0.215 .004; Trace
4.191; 4.155 .069; .060
47.027; 47.102 .582; .464
P.P.M. in liver 1 wk. of age 5 wks. of age a
( ) = number of birds. No significant mean differences. All means accompanied by a standard deviation. a - 4 weeks after Mirex withdrawal from feed. Mirex withdrawal (Table 1). Liver residues of Mirex were in the same ratio as feed levels and were markedly reduced 4 weeks after Mirex withdrawal from the feed. Increasing the level of Mirex to 400 and 800 p.p.m. and extending the feeding period over the first 5 weeks of life did not compromise the weights of the adrenal, bursa, liver, thymus
or spleen (Tables 2 and 3). The M-800 treatment significantly reduced body weight (Table 3). Antibody-mediated immunity as evaluated by production of precipitin to BSA, levels of IgG and IgM, and number of antibody producing cells (plaque forming cells, PFC) in response to SRBC was not affected by the Mirex treatment (Tables 2 and 3). Liver
TABLE 2.—Body and gland weights and antibody response of chickens receiving 400 p.p.m. of Mirex (M) from hatch to 5 weeks of age P.P.M. of Mirex M-0
M-400
Body wt. 2 wk. 4 wk.
201.4 (22) 513.2 (15)
188.5 (25) 527.2 (18)
2 weeks (6) Bursa mg./gm. Thymus mg./gm. Spleen mg./gm. Liver mg./gm.
2.87±0.75 0.43+0.11 1.70±0.73 3.33±0.25
3.01 ±0.57 0.40±0.11 1.22±0.30 3.12+0.16
1.17+0.22 3.46±0.83 20.58±3.50 80.78±43.10 603,903,2586 .026; .028; .023; .024
1.21±0.35 3.52±0.82 21.65±3.61 95.90±44.20 1277,1567,2300 19.07; 21.30; 63.15 27.14; 30.14; 23.4
5 weeks (8) Bursa Spleen Liver ixg. AbN/ml. (7) P F C / 10s spleen cells PPM in liver No significant differences. ( ) = number of birds.
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3 wks. of age (6) Bursa, mg./gm. Spleen mg./gm. Comb, mg. Hematocrit-1 wk.
M-0
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ANTIBODY-MEDIATED IMMUNITY
TABLE 3.—Body and gland weights and serum protein analysis of 5-week-old Mirex fed birds P.P.M. ( )f Mirex M-400 M-200 607b 633" 0.92±0.34 1.98+0.80 0.51±0.11 0.51+0.11 4.51±1.63 3.49±1.10
M-800 537c 647a 0.84±0.22 0.98+0.31 0.65+0.22 0.57±0.08 2.87±0.96 3.00+0.91 3.75±0.58 3.77±0.35 1.46±0.30 1.39+0.20 676±352 817.20±257 562 ±234 428.0±90 403±141 258.0±122 NM <100 >90 NM .075 Means possessing different superscripts are significantly different at the 5% level. Eight birds per mean. M-0
Body wt., gm. Bursa, mg./gm. Adrenal mg./gm. Spleen mg./gm. Total protein gm.% Albumin gm.% IgG mg.% IgM mg.% Transferrin mg.% P.P.M. in liver
T A B L E 4.-
FIG. 2. Bursal follicles from a control chick exhibiting normal basophilic cells and division of bursal follicles into medulla and cortex (x 200).
- White blood cell counts of Mirex (M) fed birds before and after the injection of four I. U. ACTH per 100 gm. body weight
TWBC x HF 2 wks. 3 wks. 4 wks.*
M-0
P.P.M. Mirex M-200
M-400
17.8 16.4 23.8
14.6 22.0 21.7
12.9 17.9 20.5
Heterophil x /0 3 4.4 5.8 2 wks. 4.9 4.9 3 wks. 12.0 4 wks.* 14.3 TWBC—Total-white-blood cell count. * = 4 I.U. ACTH/100 gm. body weight. Injected 4 hours before cell counts. No significant treatment or treatment x week differences.
2.0 4.4 12.3
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FIG. 1. Bursal follicles from a bird 3 weeks old treated with 400 p.p.m. of Mirex exhibiting pale lymphoid cells similar to cyclophosphamide-treated chicks (x 200).
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B. GLICK
TABLE 5.—Body, gland, serum protein, and antibody response of chicks receiving 500 p.p.m. of DDT (D) hatch to 5-weeks of age P.P.M. D-0 Body wt., gm. 1 wk.
2 wks. 4 wks. 2 wks. of age (12) Bursa mg. /gm. Adrenal mg./gm. Spleen mg./gm. Liver mg./gm. Comb mg.
86.78±12.14(37) 240.55±57.1 (27) 513.57±88.9 (14)
85.36±4.23 (30) 241.44±26.3 (27) 508.93±77.0(15)
2.77±0.65 0.15±0.03 1.23+0.41 33.12+12.90 79.36*±19.4
2.93±0.55 0.15+0.05 1.25+0.41 35.80* ±1.96 54.56±17.6
86.76±34.60 1883,992,1897 11.47 764.1*±55.2 311.0* ±74.1 226.54±82.2
86.65±30.6 75.81, 1106,2643 16.12 403.40±246.0
187.8± 142.0 145.44±67.7
slight reduction in the number of lymphoid cells found in the medulla of bursal follicles. By 3 weeks of age the bursal follicles of M-400 chicks revealed the presence of pale or lightly staining lymphoid cells, some dissolution of lymphoid cells and indipping of
residues of Mirex in the M-400 group varied in one experiment from 19.07-63.15 p.p.m. (Table 2) while they remained above 90 p.p.m. and below 100 p.p.m. in the other experiment (Table 3). Histologically the bursa from 2 week old M-400 treated chicks exhibited a
TABLE 6.—Body weight, gland weight, and antibody response of birds fed varying levels of DDT (D) from hatch to six weeks of age P.P.M D-400
D-800
0-1600°
98.21±58.51
75.68±24.90
108.09±53.01
81.29± 19.20
941.42 0.53±0.20 32.34±3.10 0.085±00.010 2.01 ±0.54 1.87
903.00 0.46±0.07 28.06±2.10 0.090±0.030 1.73 ±0.37 1.80
902.57 0.53±0.14 32.58+2.93 .097±0.087 2.07±0.66 1.75
D-0 5 weeks of age, 8 birds/ mean M-g. AbN/ml. 6 weeks of age, 7 birds/ mean Body, gm. Bursa, mg./gm. Liver, mg./gm. Adrenal, mg./gm. Spleen, mg./gm. Feed efficiency p.p.m. in liver P.p-DDE p,p'-DDD p,p'-DDT Total DDT
<0.10
7.1 10.2 7.0 24.2
12.0 12.0 4.3 28.3
38.9 46.6 34.7 44.5 8.3 5.6 82.0 96.7
21.7 44.9 15.2 81.8
22.5 19.0 36.5 44.3 21.0 4.6 80.0 68.3
a—Data from birds receiving 1600 p.p.m. from 1-6 weeks of age. This level lethal to chicks when initiated at hatching, see text.
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4 wks. of age M-g. AbN/ml. (8) PFC/106 spleen cells Phagocytic index (6) IgG mg.% (8) IgM mg.% (8) Transferrin mg.% (8) () = number of birds. *P < .05.
D-500
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ANTIBODY-MEDIATED IMMUNITY
epithelial cells lining the bursal folds (Figures 1 and 2). The methyl green-pyronin (MGP) stain revealed the presence of plasma cells in the spleen, caecal tonsil, and gland of Harder. The total white blood cell count and absolute number of heterophils in 2 and 3-week-old M-200 and M-400 treated chicks were similar to control values (Table 4). The three groups exhibited the normal increase in absolute number of heterophils upon challenge with 4 I.U. ACTH/100 gm. of body weight (Table 4). FIG. 3. Bursal follicles from a 3-week-old bird treated with 500 p.p.m. of DDT exhibiting dissolution of bursal follicles, vacuolation and loss of medullary cells (x 200). remaining two died in the fourth week with liver residues of 1084.8 (p,p'DDE-685.1 p.p.m. and p,p'DDD-397.6 p.p.m.) and 526.7 (p,p'DDE-301.7 p.p.m. and p,p'DDD-221.7 p.p.m.) total p.p.m. of DDT. All eight chickens exhibited ataxia within one week of death. At 1 week of age 1600 p.p.m. of DDT was added to the basal diet of 7 chicks. Within two weeks, 4 of these chicks exhibited ataxia. At 5 weeks of age, 4 weeks after receiving 1600 p.p.m. of DDT, these chickens con-
TABLE 7.—Precipitin response to BSA ("/xg. AbN/ml.) of 5-week-old DDT (D)-treated chickens (6 per mean) Feed not withheld Groups
D-0 p.p.m. D-200 p.p.m. D-400 p.p.m.
111.35b±34.90
Feed withheld during Induction and proPreinduction duction period period (4d. before (At time of BSA BSA inj.) Inj. and for next 7 days) 67.0C±44.40 (0.04±0.02) 33.03de±23.01 15.13e±8.10
Means possessing different superscripts are significantly different (p < .01). ( ) = p.p.m. of total DDT in liver from separate birds at 6 weeks of age.
118.40a±62.10 115.44b±74.50 (32.26± 14.35) 40.16d±2.00 (40.12±28.73)
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DDT. DDT, like Mirex, when administered at levels up to 800 p.p.m. failed to affect the weights of the adrenal, bursa, thymus, or spleen (Tables 5 and 6). Comb size (equal numbers of males and females) was significantly decreased (Table 5) and liver size significantly increased in one of two experiments (Tables 5 and 6). Unlike Mirex-fed birds, the DDT treated chickens exhibited significantly depressed levels of IgG and IgM (Table 5). However, the DDT-treated birds ability to produce antibody to BSA and PFC to SRBC was not significantly different from control animals (Tables 5 and 6). Administering 1600 p.p.m. of DDT at hatching was lethal to 6 of 8 chicks by two weeks of age. The
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B. GLICK
TABLE 8.—Immunoglobulin G and IgM levels (mg.%) of 6-week-old birds fed DDT (D) for 5 weeks and starved from week 5 to week 6 Feed withheld
Immunoglobulin IgG
Feed not withheld(D-O) 720.30±81.10
D-0 7O6.90±21.70
D-200 699.16±65.30
D-400 527.32±97.70
IgM
251.8±47.70
196.06±68.60
256.8±8.00
293.00± 187.30
Means not underlined by the same line are significantly different at the 1% level.
Feed Withdrawal and Pesticides. Withdrawing feed from the D-200 and D-400 birds four days before the administration of BSA (preinduction period) significantly depressed the titers below comparable controls and controls on full feed (Table 7). Also, the controls (D-0) experienced a significant reduction in antibody titers when feed was withdrawn during the preinduction period. The concentration of IgG (mg.%) was signif i-
TABLE 9.—Precipitin response (fxg. AbN/ml.) of 5-week-old Mirex fed chickens (7 per mean)
Groups M-Op.p.m.
Feed not withheld 123.3±52.50 (0.13±0.14)
M-200p.p.m.
96.18±59.40 128.85±36.50 (75.59±76.55) 122.84±77.80 (250.93+405.88)
M-400 p.p.m. M-"200" p.p.m.
Feed withheld 3 days prior to BSA injection (preinduction period)
116.35+87.8
M-"200"—Mirex added to feed in acetone. No significant differences observed. ( ) = p.p.m. Mirex in liver from two separate birds at 6 weeks of age.
cantly depressed in the D-400 group (Table 8). Withdrawing feed at the time of BSA injection and for a seven day period (induction and production periods) significantly enhanced titers in the D-0 group, did not influence titers in the D-200 birds, and significantly depressed titers in the D-400 group (Table 7). On the other hand, withdrawing feed from the M-0, M-200 and M-400 birds three days before the administration of BSA (preinduction period) did not significantly influence the preciptin levels (Table 9). Body weights were significantly depressed during starvation in all groups. For example, body weight before feed withdrawal and seven days later was 840.71 ± 61.60 and 601.85 ± 42.00 for D-0; 776.80 ± 81.61 and 547.62 ± 49.22 for D-200; and 787.50 ± 96.61 and 557.37 ± 58.20 for D-400. No mortality was experienced. Also, withdrawing feed for three days from the D-0, D-200, D-400 groups did not significantly increase the number of absolute heterophils over full fed birds. DISCUSSION We have demonstrated that the bursa is capable of regeneration following steroid induced regression during the neonatal period (Glick, 1967, 1970). The regeneration of the bursa was delayed as evidenced by a marked suppression of hemagglutinin and precipitin production at 5 weeks of age and a normal titer of these antibodies by 8 weeks of age. While steroid administration to neonatal chicks eliminated or markedly reduced the
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tained DDT-liver residues comparable to chicks fed 800 p.p.m. from hatching (Table 6). The D-500 chicks exhibited severe dissolution of bursal follicles by 3 weeks of age with vacuolation and loss of medullary cells (Figure 3). However, a second experiment revealed no obvious difference in D-500 and D-0 bursae at 3 weeks of age. The MGP stain revealed the presence of plasma cells in both the spleen and caecal tonsils.
ANTIBODY-MEDIATED IMMUNITY
Failure of excessive levels of DDT or Mirex to markedly suppress antibody-mediated immunity demonstrates that in the chicken, whose immune system has been used as a model to understand the mammalian immune system, acute exposure to DDT or Mirex will not significantly compromise humoral immunity. Our data did not include functional studies of cell-mediated immunity, but the normal histology of the thymus which controls cell-mediated immunity (Cooper et al., 1966) from DDT and Mirex birds suggests an intact cell-mediated system in the pesticide-treated birds. DDT-treated birds but not Mirex birds appeared to be sensitive to feed withdrawal. Starvation during the preinduction period significantly depressed antibody response in DDT-treated birds (D-200 and D-400) over comparable controls (D-0) and full-fed controls. Also, in this experiment the controls (D-0) starved during the preinduction period experienced a significant reduction in antibody titers when compared to full-fed controls. These data indicate that chickens exposed to high levels of DDT and faced with starvation and other possible environmental changes are less able to mount a normal antibody response. While Donaldson et al. (1968) reported mortality of DDT-fed birds after four days of starvation none of our DDT or Mirex-treated birds died during the 4-7 day starvation periods. Also, DDT and Mirex treatment did not produce physiological stress as evidenced by a lack of change in the absolute number of heterophils. There remain two avenues of study with DDT and Mirex. The first entails chronic treatment of birds with low levels of the pesticides and the assessment of the immune system of the adult and their offspring. The former has been initiated. The second approach is to administer the two pesticides to the embryo by in ovo injections or shell application and then to study the fine structure of the bursa and thymus in the embryo
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presence of bursal follicles (Glick, 1967, 1970), the alkylating agent, cyclophosphamide, did not destroy the cytoarchitecture of the bursa (Glick, 1971; Linna et al., 1972). However, cyclophosphamide administered during the first 2-3 days of the chicks life will produce agammaglobulinemia (Lerman and Weidanz, 1970; Glick, 1971; and Toivanen et al., 1972). Therefore, marked interference with bursal development during the first week of life will be reflected in varying degrees of humoral immunity suppression. Hence, the purpose of administering Mirex and DDT to neonatal chicks was to assess their potential for disrupting bursal development and future humoral immunity. Since these pesticides proved unsuccessful in initiating bursal involution during the first 2 weeks of life, one might conclude that the pesticides would not alter immunoglobulin (Ig) or antibody production. And, while this was true of Mirex it was not true for DDT. The latter significantly reduced IgG and IgM. Glick and Whatley (1966) demonstrated that o,p'DDD would suppress the precipitin response in chickens. However, the significance of this report is questionable since the DDT-analogue was administered intravenously. In rabbits receiving 200 p.p.m. of DDT in the drinking water, a significant reduction in gamma globulin was noted (Wasserman et al., 1969). These authors report individual titer values for the rabbits injected with ovalbumin and while the titers were lower in the DDT-treated rabbits by one tube my analysis of the raw data demonstrated no statistical significance. Degeneration of bursal follicles by three weeks cannot explain the Ig results in DDT-treated birds since the same regression occurred in Mirex birds without the accompanying reduction in Ig levels. The possibility that the spleen or other sites may be sensitive to increased loads of organic chemicals during the neonatal period (Glick, 1972a) might explain the DDT effect in the absence of an obvious bursal response.
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as well as antibody and cell-mediated immunity in the hatched chick. ACKNOWLEDGEMENT
REFERENCES Agthe, C., H. Garcia, P. Shubik, L. Tomatis and E. Weyon, 1970. Study of the potential carcenogenicity of DDT in the Syrian golden hamster. Proc. Soc. Exptl. Biol. Med. 134: 113-116. Cooper, M. D., R. D. A. Peterson, M. A. South and R. A. Good, 1966. The functions of the thymus system and bursa system in the chicken. J. Exp. Med. 123: 75-102. Cunningham, A. J., and A. Szenberg, 1968. Further improvements in the plaque technique for detecting single antibody producing cells. Immunology, 14: 599-600. Donaldson, W. E., T. J. Sheets and M. D. Jackson, 1968. Starvation effects on DDT residues in chick tissues. Poultry Sci. 47: 237-243. Duncan, D. B., 1955. Multiple range and multiple " F " tests. Biometrics, 11: 1-42. Fitzhugh, O. G. and A. A. Nelson, 1947. The chronic oral toxicity of DDT (2,2, bis (p-chlorophenyl-1,1,1 trichloroethane). J. Pharmac. Exp. Ther. 89: 18. Glick, B., 1967. Antibody and gland studies in cortisone and ACTH-injected birds. J. Immun. 98: 10761084. Glick, B., 1968. Serum protein electrophoresis patterns in acrylamide gel: patterns from normal and bursaless birds. Poultry Sci. 47: 807-814. Glick, B., 1970. Immunity studies in testosterone propionate injected chicks. Int. Arch. Allergy, 38: 93-103. Glick, B., 1970. The bursa of Fabricius: A central issue. BioScience, 20: 602-604. Glick, B., 1971. Morphological changes and humoral immunity in cyclophosphamide-treated chicks.
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I thank Mr. F. L. Bailey of the Allied Chemical Corporation and the Aldrich Chemical Company for generously supplying us with Mirex and DDT, respectively. The technical assistance of Sharon Brock, Diana Weigmann, and Sandra Whatley contributed to the success of this project. Special acknowledgement is made to the department of Biochemistry, Mississippi State University, Dr. Ben Barrentine, Head, and Research Chemist Jimmie Cain for the liver residue analyses.
Transplantation, 11: 433-439. Glick, B., 1972a. Cortisone, age and antibody-mediated immunity. Int. Arch. Allergy, 43: 766-771. Glick, B., 1972b. The immunobiological influence of Mirex and DDT. Poultry Sci. 51: 1861. Glick, B., and C. R. Sadler, 1961. The elimination of the bursa of Fabricius and reduction of antibody production in birds from eggs dipped in hormone solutions. Poultry Sci. 40: 185-189. Glick, B., and S. Whatley, 1966. The effect of o,p-DDD in the chicken. Experientia, 2: 179-182. Kemeny, T., and R. Tarjan, 1966. Investigations on the effects of chronically administered small amounts of DDT in mice. Experientia, 22: 748. Lerman, S. P., and W. Weidanz, 1970. The effect of cyclophosphamide on the ontogeny of the humoral immune response in chickens. J. Immunol. 105: 614-619. Linna, T. J., D. Frommel and R. A. Good, 1972. Effect of early cyclophosphamide treatment on the development of lymphoid organs and immunological functions in the chicken. Int. Arch. Allergy, 42: 20-39. Markin, G. P., J. H. Ford, J. C. Hawthorne, J. H. Spence, J. Davis and H. L. Collins, 1972. Insecticide Mirex and techniques for its monitoring. U.S.D.A. Bull. APHIS 81 3 pp. 19. May, D., and B. Glick, 1964. Weight of the bursa of Fabricius and antibody response of chicks hatched from eggs dipped in varying concentrations of testosterone propionate. Poultry Sci. 43:450-453. McGovern, J. J., A. R. Jones and A. G. Steinberg, 1955. The hematocrit of capillary blood. New England J. Med. 253: 308-312. Morgan, G. W., Jr., andB. Glick, 1972. A quantitative study of serum proteins in bursectomized and irradiated chickens. Poultry Sci. 51: 771-778. Mueller, A. P., K. Sato and B. Glick, 1971. The chicken lacrimal gland, gland of Harder, caecal tonsil, and accessory spleens as sources of antibody-producing cells. Cellular Immunology, 2: 140-152. Mueller, A. P., H. P. Wolfe and R. K. Meyer, 1960. Precipitin production in chickens. XXI. Antibody production in bursectomized chickens and in chickens injected with 19-nortestosterone on the fifth day of incubation. J. Immunol. 85: 172-179. Natt, M. P., and C. A. Herrick, 1958. A new blood diluent for counting the erythrocytes and leukocytes of the chicken. Poultry Sci. 31: 735-738. Opstad, A. M., 1959. A methyl green-pyronin stain for plasma cells in tissues. Stain Technol. 34: 293. Sato, K., and B. Glick, 1970. Are splenic plaque forming cells sensitive to heat in vitro? Life Sci. 9: 175-180. Subba Rao, D. S. V., and B. Glick, 1970. Immunosup-
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pressive action of heat in chickens. Proc. Soc. Exptl. Biol. Med., 133: 445-448. Tarjan, R., and T. Kemeny, 1969. Multigeneration studies of DDT in mice. Fd. Cosmet. Toxicol. 7: 215-222. Thompson, J. F., 1971. Micro methods for chlorinated hydrocarbon pesticides and metabolites for human tissue and excreta, section 5A (2A). In: Manual of Analytical Methods. Published by Perrine Laboratories, Florida. Toivanen, P., A. Toivanen, T. J. Linna and R. A. Good, 1972. Ontogeny of bursal function in chick-
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ens. II. Post embryonic stem cell for humoral immunity. J. Immunol. 109: 1071-1080. Warner, N. L., A. Szenberg and F. M. Burnet, 1962. The immunological role of different lymphoid organs in the chicken. I. Dissociation of immunological responsiveness. Aust. J. Exp. Biol. 40: 373-388. Wasserman, M., D. Wasserman, Z. Gershon and L. Zellermayer, 1969. Effects of organochlorine insecticides on body defense systems. Ann. New York Acad. Sci. 160: 393-401. Woodwell, G. B., 1967. Toxic substances and ecological cycles. Sci. Amer. 216: 24-36.
D . C. BORRON, M . G. MCCARTNEY AND H . L . FULLER
Department of Poultry Science, University of Georgia, Athens, Georgia 30602 (Received for publication November 15, 1973)
ABSTRACT These studies were undertaken to determine the effects of restricted energy feeding during the growing period upon the subsequent reproductive performance of Large White turkey females. Energy intake was limited to two-thirds of that voluntarily consumed by birds receiving practical-type growing diets ad libitum. Dietary treatments were imposed from either 8 to 32, 12 to 24, or 12 to 28 weeks of age under either natural increasing, natural decreasing, or constant eight-hour daylength. Data were collected on the number of days after the onset of stimulatory light to first egg and to 50 percent egg production, and on body weight at the end of the restriction period, at 50 percent egg production, and at the end of the reproductive period. Data were also collected on shank length, egg production, egg weight, fertility, hatchability, number of poults per hen, and carcass composition at the end of the restriction period and at 50 percent egg production. Females grown on restricted energy diets were less fat, had shorter shanks and weighed less at the end of the restriction period. Carcass fat, however, was not significantly affected at the time of 50 percent egg production. Only a slight delay in sexual maturity was noted in those birds grown on restricted energy diets. Restricting energy during the growing period did not affect reproductive performance. POULTRY SCIENCE 53: 1485-1493, 1974
T
URKEY poults grown for breeding stock are normally provided the same diets fed market birds until just prior to the time they are brought into egg production. Growing and finishing diets are designed to give maximum growth and finish at market age and, consequently, may not be optimum for growing turkeys that will be used for breeders for the production of hatching eggs. Several feeding programs are being used by producers of hatching eggs to delay sexual maturity and to improve the reproductive
performance of the chicken. Quantitative feed restriction during the rearing period has been shown to delay sexual maturity and improve reproductive performance of both broiler breeder and egg-type chickens (Isaacks et al., 1960; Hollands and Gowe, 1961; Fuller and Dunahoo, 1962; and Strain et al., 1965). Low protein diets have also been found to be effective in delaying maturity and improving reproductive performance of the chicken (Wright et al., 1968). Dietary energy restriction also has been shown to
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The Effects of Restricted Energy Feeding During the Growing Period on the Reproductive Performance of Turkey Breeder Females