Effect of Vitamin E and A on Humoral Immunity and Phagocytosis in E. coli Infected Chicken ROBERT P. TENGERDY and JANE C. BROWN Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523
ABSTRACT Dietary supplementation of either vitamin E (300 mg./kg. diet) or vitamin A (60,000 I.U./kg. diet) significantly reduced E. coli caused mortality, but the combination of the two vitamins did not. Protection was attributed to increased antibody production and increased phagocytosis, although neither factor alone gave a significant correlation with mortality. Vitamin E level significantly increased especially in the spleen of supplemented chicks, but vitamin A suppressed this increase, partially explaining the lack of protection in vitamin E and A supplemented chicks. Poultry Science 56:957-963, 1977
This report is a sequel to a previous report on this topic (Tengerdy and Nockels, 1975). Although a protective effect of either vitamin E or A against E. coli infection was clearly established earlier, neither the mechanism of the effect nor the puzzling antagonistic effect of vitamins E and A, when applied together, could be explained. In this report we attempted to throw some light on these points. MATERIALS AND METHODS One day old broiler chicks, males and females, from a disease free commercial stock, were randomly assigned into nine dietary groups as indicated in Table 1. The diet, vitamin supplements, and handling of chicks were the same as reported previously (Tengerdy and Nockels, 1975). The composition of the Colorado State University standard chicken diet is given in Table 2. The chicks were housed in wire floored, electrically heated battery brooders, 15 chicks per pen, each battery being arranged so that accidental food spillage would not introduce a higher level of vitamin into a lower pen. The batteries and water supply was kept clean throughout the experiment. The E. coli strain was a highly pathogenic rifampin resistant mutant. Virulence of this strain was maintained by serial passage in two day old chicks. The chicks were sacrificed just before dying from the infection, and the liver containing a high concentration of E. coli was used as a source of inoculum for trypticase soy
broth (TSB) cultures, or the liver was quickly frozen for storage. The inoculum from the frozen liver needed only one passage after even prolonged storage to regain the high previous virulence, within a ± 5-fold variation in the LD 5 0 dose. The experiment protocol for the main experiment, experiment 1, is given in Table 3. Half of the chicks in groups 2, 4, 6, and 10 were immunized with 2.0 X 10 6 E. coli live cells, grown in TSB for 18 hours, at age seven days, and the survivors were challenged with 2.0 X 10 9 cells at 21 days. The rest of the chicks, excluding absolute controls, were challenged with 2.0 X 10 8 cells at 21 days. The above doses were the approximate L D 5 0 doses for immunized or non-immunized chicks determined in a separate experiment. All E. coli infections were I.V. into the wing vein. In a second follow-up experiment (Table 5) killed E. coli cells were used for immunization at day seven to avoid the about 30% loss of chicks resulting from live immunization. The immunizing dose was 2.0 X 10 7 cells. The challenge dose on day 21 was 3.0 X 10 7 live cells for the non-immunized chicks, and 3.0 X 10 8 cells for immunized chicks, the L D S 0 doses at the time of the test. A blood clearance test, as a measure of phagocytosis, was performed on day 21 with five chicks in each group receiving E, coli. Samples were taken from the wing vein opposite to the site of infection at 0, 1, 2, and 3 hours post infection. The blood was diluted in Alsever solution and plated on nutrient agar plates for counting. Hemagglutination titers
957
Downloaded from http://ps.oxfordjournals.org/ at Kokusai Hoken Keikakugaku (UNIV OF TOKYO) on June 10, 2015
(Received for publication .October 15, 1976)
958
T E N G E R D Y A N D BROWN T A B L E 1. — Vitamin
supplementation
of dietary
groups
Vitamin added mg./kg. or I.U./kg
Total vitamin level in diet b y assay* mg./kg. or I.U./kg
Diet code
150 300
150 30,000 60,000 30,000 60,000 30,000 60,000
150 150 300 3Q0
NA NA 42,850 74,075 50,000 70,330 41,000 68,000
215 328 NA 210 212 305 315
• C o u r t e s y of Dr. C. R. A d a m s , Hoffmann-LaRoche, I n c o r p o r a t e d .
T A B L E 2.—Composition
Ingredients G r o u n d corn G r o u n d milo Soybean meal ( 4 4 protein) Fish meal (70 protein) Meat and b o n e meal ( 5 0 protein) D e h y d r a t e d alfalfa ( 1 7 protein) G r o u n d limestone Phosphate supplies ( P = 1 8 Ca=24) Salt Animal fat Vitamin mix* Trace mineral m i x * * Methionine Total Calculated analysis: Protein, % Fat, % Fiber, % Calcium, % Phosphorus (Total) Phosphorus (Available) Lysine Meth and Cys Energy calories/lb.: Productive C:P ratio Metabolizable C:P ratio
of feed. Colorado State University; Department C. S. U. broiler-roaster rations Broiler starter 23%
of Avian
Broiler finisher 20%
Science.
Broiler finisher 18%
31.00 22.60 30.00 3.00 5.00 2.50 0.70 0.50 0.40 4.00 0.25 0.05 0.05
31.00 30.60 22.50 2.50 5.00 2.50 0.60 0.50 0.50 4.00 0.25 0.05 0.05
31.00 33.10 24.30
100.00
100.00
100.00
23.00 6.40 3.80 1.00 0.77 0.54 1.40 0.80
20.10 6.50 3.50 1.00 0.72 0.50 1.10 0.71
18.10 6.40 3.60 0.90 0.68 0.45 0.95 0.64
1020.0 44:1 1347.0 58:1
1047.0 52:1 1384.0 69:1
2.50 2.50 0.60 1.20 0.50 4.00 0.25 0.05 0.05
1047.0 58:1 1384.0 77:1
• V i t a m i n m i x supplies t h e following per p o u n d of r a t i o n : V i t a m i n A 2 4 0 0 I.U.; V i t a m i n D 4 0 0 I.C.U.; Vitamin K .15 mg.; Riboflavin 2 m g . ; Niacin 10 mg.; P a n t o t h e n i c acid 2 mg.; and Choline 1 4 8 mg. **Trace mineral m i x supplies t h e following per p o u n d of feed: Mn 50 p.p.m.; Fe 50 p . p . m . ; Cu 5.0 p . p . m . ; C o 0.5 p.p.m.; I 1.5 p . p . m . ; and Zn 50 p . p . m .
Downloaded from http://ps.oxfordjournals.org/ at Kokusai Hoken Keikakugaku (UNIV OF TOKYO) on June 10, 2015
Absolute control Infection c o n t r o l E, E2 A, A2 E,A, E,A2 E2A, E2A2
VITAMINS E AND A AND IMMUNITY
959
TABLE 3. — Experimental protocol for experiment 1 Number of chicks treated Started at
Group
Group code Absolute control (AC) Control IC
3 4
E, IE2
C
5 6
E2 A,
I A2 A,
7 8 9 10
E,A, E,A 2 E 2 A, IE2A, E2A2
Total
30 30 30 30 30 30 30 30 30 30 30 30 30 30 420
30
30
30
30
120
21
Phagocytic study at day 21
HA titer at 28
day 35
5 5
30 11 17 16 17 25 16 19 26 15 19 19 14 22
30 10 17 16 17 25 15 19 26 15 19 18 12 22
40
266
261
20 30 30 22 30 28 23 30 26 29 30 20 30
5 5
348
5 5 5 5
Each group contained randomly assigned replicates of 15 one day old broiler chicks of both sexes per brooder compartment. I stands for immunized.
(HA) at seven and 14 days post infection were measured by the microtitration technique described previously, using an E. coli LPS antigen coated on formalinized sheep red blood cells (Tengerdy and Nockels, 1975). Statistical analysis, analysis of variance (for all chicks) and Tukey's multiple range test for comparison of treatment means for the HA data, as well as the correlation coefficients for comparing mortality, HA titers and phagocytosis was performed by standard methods (Snedecor and Cochran, 1967). RESULTS The results of experiment 1 are summarized in Table 4. The main conclusions of previous experiments (Tengerdy and Nockels, 1975) were again corroborated; vitamin E or vitamin A helps to reduce mortality resulting from E. coli infection, but the combination of the two vitamins is less effective. Phagocytosis aided by humoral antibody appears to be a contributing factor to the increased immunity. Vitamin E and A enhanced both humoral immunity and phagocytosis, but the overall correlation between mortality and phagocytosis or hemagglutination (HA) titers was not significant. It was particularly puzzling that while early
phagocytosis was most stimulated in the E 2 A 2 group, (c.f. Table 1) and HA titers were reasonably high, mortality remained relatively high. The vitamins affected only the early stages of phagocytosis. The maximum effect in blood clearance was found at one hour. At two or three hours the differences between control and treatment groups became less and less, perhaps because by three hours a slow rise in the number of viable E. coli cells was observed in all groups. The number of E. coli cells recovered two or three hours after injection varied widely, apparently reflecting the individual resistance of the chicks, but making statistical evaluations impossible. Phagocytosis was three-four times faster in all immunized birds, demonstrating the efficacy of immune clearance over non-immune clearance. The effect of vitamin E and A on immune clearance was, however, about the same magnitude as on non-immune clearance, suggesting that other, hitherto unknown, factors must play a role in the enhanced protection. Hoping to get more precise phagocytic data, and to check the reproducibility of these data, a second experiment was conducted with the critical groups as shown in Tables 5 and 6. Although the conditions of this second experiment were slightly different, since killed E. coli was used for immunization, and the virulence
Downloaded from http://ps.oxfordjournals.org/ at Kokusai Hoken Keikakugaku (UNIV OF TOKYO) on June 10, 2015
1 2
day 0
Immunized at day 7
Infected at dav
TENGERDY AND BROWN
960
TABLE 4. — Mortality and blood clearance (phagocytosis) in experiment 1 Number of E. coli cleared from blood Group
HA log2 titer
Group code
3 4
E 2
5 6
A, IA, A2
E,A, E,A 2 E*A, IE2A2 E2A2
7 8 9 10
14 day
7 day
2.1 ± 0.9 a 0.5 ± 0 . 3 A 3.5 ± 1.6b 1.2 ± 0.7B 3.6 ± 1.8b 0.8 ± 0.4B
6.1 ± 3.0C 1.8 + 0.7^
2.2 ± 0.9 10.7 ± 1.0a 5.0 ± 0.5A 5.2 ± 0 . 6 A 14.1 ± 0.8b 7.4 ± l.OB 5.3 ± 0.6A 13.2±0.8b 6.5 ±0.7B 6.0 ± 0.8A 6.2 ± 0.9A 5.4 ± l.OA 11.2 ±0.8b 5.8 ±0.5A
2.1 12.8 6.0 6.0 13.5 8.3 7.2 14.8 7.8 6.2 6.2 6.0 13.2 6.2
± 1.0 ± 0.8 a ± 1.0 A ± 0.9A + 0.7 a ± 0.6 B + 0.9B ± 0.7 a ± l.OB ±0.5A ± 0.8A ± 0.7A ± 0.8 a ± 0.7A
Different letters indicate significant differences among groups at p
ABSTRACT Dietary supplementation of either vitamin E (300 mg./kg. diet) or vitamin A (60,000 I.U./kg. diet) significantly reduced E. coli caused mortality, but the combination of the two vitamins did not. Protection was attributed to increased antibody production and increased phagocytosis, although neither factor alone gave a significant correlation with mortality. Vitamin E level significantly increased especially in the spleen of supplemented chicks, but vitamin A suppressed this increase, partially explaining the lack of protection in vitamin E and A supplemented chicks. Poultry Science 56:957-963, 1977
This report is a sequel to a previous report on this topic (Tengerdy and Nockels, 1975). Although a protective effect of either vitamin E or A against E. coli infection was clearly established earlier, neither the mechanism of the effect nor the puzzling antagonistic effect of vitamins E and A, when applied together, could be explained. In this report we attempted to throw some light on these points. MATERIALS AND METHODS One day old broiler chicks, males and females, from a disease free commercial stock, were randomly assigned into nine dietary groups as indicated in Table 1. The diet, vitamin supplements, and handling of chicks were the same as reported previously (Tengerdy and Nockels, 1975). The composition of the Colorado State University standard chicken diet is given in Table 2. The chicks were housed in wire floored, electrically heated battery brooders, 15 chicks per pen, each battery being arranged so that accidental food spillage would not introduce a higher level of vitamin into a lower pen. The batteries and water supply was kept clean throughout the experiment. The E. coli strain was a highly pathogenic rifampin resistant mutant. Virulence of this strain was maintained by serial passage in two day old chicks. The chicks were sacrificed just before dying from the infection, and the liver containing a high concentration of E. coli was used as a source of inoculum for trypticase soy
broth (TSB) cultures, or the liver was quickly frozen for storage. The inoculum from the frozen liver needed only one passage after even prolonged storage to regain the high previous virulence, within a ± 5-fold variation in the LD 5 0 dose. The experiment protocol for the main experiment, experiment 1, is given in Table 3. Half of the chicks in groups 2, 4, 6, and 10 were immunized with 2.0 X 10 6 E. coli live cells, grown in TSB for 18 hours, at age seven days, and the survivors were challenged with 2.0 X 10 9 cells at 21 days. The rest of the chicks, excluding absolute controls, were challenged with 2.0 X 10 8 cells at 21 days. The above doses were the approximate L D 5 0 doses for immunized or non-immunized chicks determined in a separate experiment. All E. coli infections were I.V. into the wing vein. In a second follow-up experiment (Table 5) killed E. coli cells were used for immunization at day seven to avoid the about 30% loss of chicks resulting from live immunization. The immunizing dose was 2.0 X 10 7 cells. The challenge dose on day 21 was 3.0 X 10 7 live cells for the non-immunized chicks, and 3.0 X 10 8 cells for immunized chicks, the L D S 0 doses at the time of the test. A blood clearance test, as a measure of phagocytosis, was performed on day 21 with five chicks in each group receiving E, coli. Samples were taken from the wing vein opposite to the site of infection at 0, 1, 2, and 3 hours post infection. The blood was diluted in Alsever solution and plated on nutrient agar plates for counting. Hemagglutination titers
957
Downloaded from http://ps.oxfordjournals.org/ at Kokusai Hoken Keikakugaku (UNIV OF TOKYO) on June 10, 2015
(Received for publication .October 15, 1976)
958
T E N G E R D Y A N D BROWN T A B L E 1. — Vitamin
supplementation
of dietary
groups
Vitamin added mg./kg. or I.U./kg
Total vitamin level in diet b y assay* mg./kg. or I.U./kg
Diet code
150 300
150 30,000 60,000 30,000 60,000 30,000 60,000
150 150 300 3Q0
NA NA 42,850 74,075 50,000 70,330 41,000 68,000
215 328 NA 210 212 305 315
• C o u r t e s y of Dr. C. R. A d a m s , Hoffmann-LaRoche, I n c o r p o r a t e d .
T A B L E 2.—Composition
Ingredients G r o u n d corn G r o u n d milo Soybean meal ( 4 4 protein) Fish meal (70 protein) Meat and b o n e meal ( 5 0 protein) D e h y d r a t e d alfalfa ( 1 7 protein) G r o u n d limestone Phosphate supplies ( P = 1 8 Ca=24) Salt Animal fat Vitamin mix* Trace mineral m i x * * Methionine Total Calculated analysis: Protein, % Fat, % Fiber, % Calcium, % Phosphorus (Total) Phosphorus (Available) Lysine Meth and Cys Energy calories/lb.: Productive C:P ratio Metabolizable C:P ratio
of feed. Colorado State University; Department C. S. U. broiler-roaster rations Broiler starter 23%
of Avian
Broiler finisher 20%
Science.
Broiler finisher 18%
31.00 22.60 30.00 3.00 5.00 2.50 0.70 0.50 0.40 4.00 0.25 0.05 0.05
31.00 30.60 22.50 2.50 5.00 2.50 0.60 0.50 0.50 4.00 0.25 0.05 0.05
31.00 33.10 24.30
100.00
100.00
100.00
23.00 6.40 3.80 1.00 0.77 0.54 1.40 0.80
20.10 6.50 3.50 1.00 0.72 0.50 1.10 0.71
18.10 6.40 3.60 0.90 0.68 0.45 0.95 0.64
1020.0 44:1 1347.0 58:1
1047.0 52:1 1384.0 69:1
2.50 2.50 0.60 1.20 0.50 4.00 0.25 0.05 0.05
1047.0 58:1 1384.0 77:1
• V i t a m i n m i x supplies t h e following per p o u n d of r a t i o n : V i t a m i n A 2 4 0 0 I.U.; V i t a m i n D 4 0 0 I.C.U.; Vitamin K .15 mg.; Riboflavin 2 m g . ; Niacin 10 mg.; P a n t o t h e n i c acid 2 mg.; and Choline 1 4 8 mg. **Trace mineral m i x supplies t h e following per p o u n d of feed: Mn 50 p.p.m.; Fe 50 p . p . m . ; Cu 5.0 p . p . m . ; C o 0.5 p.p.m.; I 1.5 p . p . m . ; and Zn 50 p . p . m .
Downloaded from http://ps.oxfordjournals.org/ at Kokusai Hoken Keikakugaku (UNIV OF TOKYO) on June 10, 2015
Absolute control Infection c o n t r o l E, E2 A, A2 E,A, E,A2 E2A, E2A2
VITAMINS E AND A AND IMMUNITY
959
TABLE 3. — Experimental protocol for experiment 1 Number of chicks treated Started at
Group
Group code Absolute control (AC) Control IC
3 4
E, IE2
C
5 6
E2 A,
I A2 A,
7 8 9 10
E,A, E,A 2 E 2 A, IE2A, E2A2
Total
30 30 30 30 30 30 30 30 30 30 30 30 30 30 420
30
30
30
30
120
21
Phagocytic study at day 21
HA titer at 28
day 35
5 5
30 11 17 16 17 25 16 19 26 15 19 19 14 22
30 10 17 16 17 25 15 19 26 15 19 18 12 22
40
266
261
20 30 30 22 30 28 23 30 26 29 30 20 30
5 5
348
5 5 5 5
Each group contained randomly assigned replicates of 15 one day old broiler chicks of both sexes per brooder compartment. I stands for immunized.
(HA) at seven and 14 days post infection were measured by the microtitration technique described previously, using an E. coli LPS antigen coated on formalinized sheep red blood cells (Tengerdy and Nockels, 1975). Statistical analysis, analysis of variance (for all chicks) and Tukey's multiple range test for comparison of treatment means for the HA data, as well as the correlation coefficients for comparing mortality, HA titers and phagocytosis was performed by standard methods (Snedecor and Cochran, 1967). RESULTS The results of experiment 1 are summarized in Table 4. The main conclusions of previous experiments (Tengerdy and Nockels, 1975) were again corroborated; vitamin E or vitamin A helps to reduce mortality resulting from E. coli infection, but the combination of the two vitamins is less effective. Phagocytosis aided by humoral antibody appears to be a contributing factor to the increased immunity. Vitamin E and A enhanced both humoral immunity and phagocytosis, but the overall correlation between mortality and phagocytosis or hemagglutination (HA) titers was not significant. It was particularly puzzling that while early
phagocytosis was most stimulated in the E 2 A 2 group, (c.f. Table 1) and HA titers were reasonably high, mortality remained relatively high. The vitamins affected only the early stages of phagocytosis. The maximum effect in blood clearance was found at one hour. At two or three hours the differences between control and treatment groups became less and less, perhaps because by three hours a slow rise in the number of viable E. coli cells was observed in all groups. The number of E. coli cells recovered two or three hours after injection varied widely, apparently reflecting the individual resistance of the chicks, but making statistical evaluations impossible. Phagocytosis was three-four times faster in all immunized birds, demonstrating the efficacy of immune clearance over non-immune clearance. The effect of vitamin E and A on immune clearance was, however, about the same magnitude as on non-immune clearance, suggesting that other, hitherto unknown, factors must play a role in the enhanced protection. Hoping to get more precise phagocytic data, and to check the reproducibility of these data, a second experiment was conducted with the critical groups as shown in Tables 5 and 6. Although the conditions of this second experiment were slightly different, since killed E. coli was used for immunization, and the virulence
Downloaded from http://ps.oxfordjournals.org/ at Kokusai Hoken Keikakugaku (UNIV OF TOKYO) on June 10, 2015
1 2
day 0
Immunized at day 7
Infected at dav
TENGERDY AND BROWN
960
TABLE 4. — Mortality and blood clearance (phagocytosis) in experiment 1 Number of E. coli cleared from blood Group
HA log2 titer
Group code
3 4
E 2
5 6
A, IA, A2
E,A, E,A 2 E*A, IE2A2 E2A2
7 8 9 10
14 day
7 day
2.1 ± 0.9 a 0.5 ± 0 . 3 A 3.5 ± 1.6b 1.2 ± 0.7B 3.6 ± 1.8b 0.8 ± 0.4B
6.1 ± 3.0C 1.8 + 0.7^
2.2 ± 0.9 10.7 ± 1.0a 5.0 ± 0.5A 5.2 ± 0 . 6 A 14.1 ± 0.8b 7.4 ± l.OB 5.3 ± 0.6A 13.2±0.8b 6.5 ±0.7B 6.0 ± 0.8A 6.2 ± 0.9A 5.4 ± l.OA 11.2 ±0.8b 5.8 ±0.5A
2.1 12.8 6.0 6.0 13.5 8.3 7.2 14.8 7.8 6.2 6.2 6.0 13.2 6.2
± 1.0 ± 0.8 a ± 1.0 A ± 0.9A + 0.7 a ± 0.6 B + 0.9B ± 0.7 a ± l.OB ±0.5A ± 0.8A ± 0.7A ± 0.8 a ± 0.7A
Different letters indicate significant differences among groups at p
