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The Use of Sulfonamides in the Control of Pullorum Disease
The Use of Sulfonamides in the Control of Pullorum Disease C. A. BOTTORFF AND J. S. KlSER Lederle Laboratories Division, American Cyanamid Company, Pearl River, New York
O TUDIES on the use of sulfonamides in ^ reducing mortality from pullorum disease were reported by Severens, Roberts and Card (1945). They found that sulfadiazine and sulfamerazine-treated birds showed a reduction in mortality from 70% to 96% in comparison to untreated corir trol birds. Sulfamerazine was as effective in 0.5% level in the feed as a 2.0% level of sulfadiazine. Hens that had been infected at 1 day of age and" treated with sulfadiazine and sulfamerazine did not react to the pullorum agglutination test at 9 months of age. Mullen (1946) reported a difference of 14% in mortality with turkeys treated wifh 0.5% sulfamerazine in comparison to untreated controls in eleven flocks totaling 22,965 turkey poults. The results of these investigators showed that those sulfonamides which produced a relatively high blood level without toxicity gave promise of being effective chemotherapeutic agents for the treatment and control of pullorum disease. Three experiments were conducted to study the effect of sulfadiazine, sulfamerazine and sulfamethazine in the control of pullorum. Sulfamethazine was included because it gave blood levels equivalent to sulfamerazine at similar dosages and preliminary in vitro studies showed it to be effective against some gram negative organisms.
U. S. Pullorum-Clean flock were used in all experiments. The chicks were artificially infected, orally, with a broth culture of S. pullorum when they were transferred from the chick boxes to battery brooders. Feed containing the sulfonamides was placed in the feed troughs before the chicks were placed in the battery so that they had access to it immediately following inoculation. The sulfa drugs were administered in the feed for 5 days in experiment 1, for 10 days in experiment 2 and for 5 days in experiment 3. The chicks in all three experiments were weighed at 21 days of age and the average weight calculated. All chicks that died were examined bacteriologically for the presence of S. pullorum. The chicks in experiments 1 and 2 were inoculated with 500 million viable 5. pullorum organisms, while in experiment 3 the dose was reduced to approximately 5 million viable organisms per chick. In experiments 1 and 2 the amount of feed consumed during medication was determined and the average amount of drug intake per chick per day was computed. In experiment 1 the blood levels of each drug in various concentrations in the feed were secured at 24 hours, 48 hours and 96 hours after medication was started.
PROCEDURE Day old New Hampshire chicks from a 335
EXPERIMENT 1
Sulfadiazine was used in higher levels than sulfamerazine and sulfamethazine because Severns, Ripberts and' Card
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(Received for publication December 7, 1946)
336
C. A. BOTTORFF AND J . S. KlSER TABLE 1.—Mortality and average weight pullorum disease experiment 1 No. chicks started
Sulfamethazine
0.75 0.50 0.25
Sulfadiazine
Sulfamerazine
Mortality 3 weeks
Average wt. in grams
No.
Percent
50 50 50
15 26 31
30 52 62
121 120 119
1.50 1.0 0.75
50 50 50
14 25 22
28 50 44
128 132 127
0.75 0.50 0.25
50 50 50
26 32 32
52 64 64
119 125 124
Infected, Untreated
—
50
47
94
84
Uninfected, Untreated
—
25
0
0
135
(1945) found that 2.0% sulfadiazine was required to obtain the same result as 0.5% of sulfamerazine. The drug was fed for 5 days. The highest mortality rate occurred during the first ten days in the infected controls. The mortality in the treated groups was negligible during the treatment period. When the drug was withdrawn from the diet, the mortality rate began to increase rapidly after five days, leveling off between the 17th to 19th day of age. Sulfamethazine showed a lower percentage of loss though there were not sufficient birds to show a significant difference. Sulfadiazine at 0.75% level gave as good protection as did sulfa-
merazine and sulfamethazine at the same levels, indicating that the three drugs might be of equal value in the treatment of pullorum disease. There was little difference in the average weights of the treated birds at 21 days of age. For mortality and average weights, see Table 1. Blood levels were obtained on a representative number of chicks from each group of treated birds. Both the free and total amounts of the drug were determined. The results of these tests are shown in Table 2. The amount of the conjugated drug was calculated by subtracting the percent of free drug from the percent of
TABLE 2.—Blood levels in milligrams percent pullorum disease experiment 1 24 hours Drug
% drug in mash
Free
Conjugated
48 hours Free
96 hours
Conjugated
Free
Conjugated
Sulfamethazine
0.75 0.50 0.25
19.13 12.90 8.90
1.33 0.00 0.00
19.13 . 15.57 9.34
2.22 0.00 0.00
28.47 16.90 11.12
0.44 0.00 0.00
Sulfamerazine
0.75 0.50 0.25
16.90 • 18.16 10.98
0.42 0.00 0.85
22.81 18.16 10.14
0.00 0.00 0.00
31.68 24.08 16.05
4.32 1.26 0.00
Sulfadiazine
1.50 1.00 0.75
28.00 24.80 27.20
1.60 3.20 1.60
40.40 30.40 23.20
8.00 10.80 8.00
39.20 31.20 25.60
8.00 7.60 6.20
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Percent drug in mash
Drug
337
U S E OF SULFONAMIDES IN CONTROL OF PULLORUM DISEASE
trols was 86% during the first 10 days, but it was 6% to 16% in the groups treated with high sulfa drug levels and 14% to 32% in those groups treated with low sulfa drug levels. In three to five days after the drug was withdrawn from the diet there was an increase in mortality that levelled off by the 20th to 21st day
TABLE 3.—Mortality and average weight pullorum disease experiment no. 2 Percent drug in mash
No. chicks started
Sulfamethazine
0.75 0.50 0.25
Sulfadiazine
Sulfamerazine
Mortality 3 weeks -No.
Percent
Average wt. in grams at 21 days
50 50 50
21 21 31
42 42 62
111 112 117
0.75 0.50 0.25
50 50 50
21 23 26
42 46 56
122 126 113
0.75 0.50 0.25
50 50 50
10 21 31
20 42 62
119 119 122
Infected, Untreated
—
50
47
94
93
Uninfected, Untreated
—
29
1*
2
177
Uninfected, Sulfamethazine
0.75
28
0
0
145
Uninfected, Sulfadiazine
0.75
28
0
0
176
Uninfected, Sulfamerazine
0.75
28
0
0
159
Drug
* Chick showed unhealed navel. 5. pullorum not isolated. EXPERIMENT 2
\
Due to the high mortality of the treated birds in experiment 1, it was decided to extend the treatment from 5 days to 10 days in experiment 2. Sulfadiazine was used at the same level as sulfamerazine and sulfamethazine to secure comparative values of the three drugs. In addition, to detect possible toxicity, three uninfected control groups were given sulfamethazine, sulfadiazine and sulfamerazine at a level of 0.75% in the feed for 10 days. The mortality rate in the infected con-
There was little difference in mortality at comparative levels in experiments 1 and 2. There was no significant difference in weight gains between any two groups of the treated infected birds. The treated infected birds made better weight gains than the infected untreated birds. In the treated uninfected groups the sulfadiazine-treated group made weight gains equivalent to the uninfected untreated controls. The sulfamethazine and sulfamerazine-treated groups made better weight gains than the infected treated
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the total drug. There was little or no conjugation of sulfamethazine and sulfamerazine, but at high levels sulfadiazine showed some conjugation. Presumably the conjugated drug is inactive. This indicated that lower levels of sulfadiazine might give better protection than was secured at a 0.75% level.
338
C. A. BOTTORFF AND J. S. KlSEK
TABLE ABLE 4.—Average 4
amount drug consumed per chick in the controls, it was decided to reduce per day pullorum disease experiments 1 and 2 Percent of drug in mash
Drug
Drug Used Mg./Ch ick/Day Experi- Experiment 1 ment 2
0.75 0.50 0.25
44 28 14
57 35 17
Sulfadiazine
1.50 1.00 0.75 0.50 0.25
81 58 45
•—
— —
48 35 18
0.75 0.50 0.25
40 26 14
41 35 16
Sulfamerazine
groups but less than the uninfected untreated controls. The mortality and average weights for experiment 2 are given in Table 3. In experiments 1 and 2 the amount of feed consumed during medication was determined and the average amount of drug consumed per chick per day was computed. The results are given in Table 4. EXPERIMENT 3
Because of the extremely high mortality
TABLE 5.—Mortality and average weight pullorum disease experiment 3 Mortality 3 weeks No.
Percent
Average wt. in grams at 21 days
104 105 105
13 16 15
12.4 15.2 14.3
153 143 148
0.75 0.50 0.25
105 105 105
9 10 18
8.6 9.5 17.1
149 150 142
0.75 0.50 0.25
104 105 105
9 8 13
8.6 7.6 12.4
145 152 158
Infected, Untreated
—
105
67
64.0
119
Uninfected, Untreated
—
74
1.4
188
Percent drug in mash
No. chicks started
Sulfamethazine
0.75 0.50 0.25
Sulfadiazine
Sulfamerazine
Drug
* Chick showed unhealed navel. S. pullorum not isolated.
1*
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Sulfamethazine
the infective dose in an effort to approach more nearly the average mortality that occurs in natural field outbreaks. This was accomplished by reducing the infective dose from 500 million to 5 million viable S. 'pullorum organisms per chick. Since no benefit was secured by the longer period of treatment in experiment 2, it was decided to treat the chicks for only 5 days. The number of chicks per group was increased to 105 chicks to try to reduce any errors that might occur where smaller lots of chicks were used. The mortality in the treated birds ranged from 8% to 12% at the high sulfa drug levels and from 12% to 17% at the low sulfa drug levels as compared to 64% in the untreated controls. Here again the mortality was checked during the period of treatment and the majority of deaths occurred between the 9th and 18th days. There was no significant difference in the weight gains of any of the treated groups. The mortality and average weights are given in Table 5. When the survivors of this group were blood tested at 12 weeks of age by the
USE OF SULFONAMIDES IN CONTROL OF PULLORUM DISEASE
rapid whole blood test for pullorum disease, approximately 90% of the birds showed a positive reaction to the test. DISCUSSION
No additional benefits appeared to be secured by feeding the drugs for more than five d a y s a t any one time. The infection was checked within five days if there was adequate consumption of the sulfonamide feed mixture.
In the dosages employed no toxic effects were observed or was there any significant effect on the weight gains of birds treated by any of the three drugs. In all experiments there was a minimum of 30% to a maximum of 56% reduction in mortality. SUMMARY 1. Sulfadiazine, sulfamethazine and sulfamerazine were equally effective in the reduction of mortality from pullorum disease in baby chicks. 2. A minimum of 30% to a maximum of 56% reduction in mortality occurred in treated groups as compared to untreated controls. 3. No significant difference in weight gains was found after 21 days in the treated groups of birds. 4. No evidence of toxicity was noted. 5. Ninety percent of surviving chicks reacted to the rapid whole blood test for pullorum disease at 12 weeks of age. REFERENCES
Mullen, F. E., 1946. Sulfamerazine as a prophylactic in pullorum disease in poults. Jour. Am. Vet. Med. Assoc. 108: 163-164. Severns, J. M., E. Roberts, and L. E. Card, 1945. The effect of sulfonamides in reducing mortality from pullorum disease. Poultry Sci. 24: 155-158.
Downloaded from http://ps.oxfordjournals.org/ at Uniwersytet Warszawski Biblioteka Uniwersytecka on May 12, 2015
There was no significant difference between sulfadiazine, sulfamethazine and sulfamerazine in their effectiveness in the reduction of mortality under the conditions of these experiments. The first two experiments failed to give as good results as experiment 3 due to the overwhelming infective dose of S. pullorum organisms. When the infective dose per chick was reduced so that it would more nearly approximate the degree of infection that occurs in a severe natural outbreak, better protection was secured. It is possible that this mortality could be reduced further by using two or three treatments of three or four days duration with four to five days between treatments. This time interval is based on the recurrence of mortality beginning five days after withdrawal of the drugs from the diet in all three experiments.
339
O TUDIES on the use of sulfonamides in ^ reducing mortality from pullorum disease were reported by Severens, Roberts and Card (1945). They found that sulfadiazine and sulfamerazine-treated birds showed a reduction in mortality from 70% to 96% in comparison to untreated corir trol birds. Sulfamerazine was as effective in 0.5% level in the feed as a 2.0% level of sulfadiazine. Hens that had been infected at 1 day of age and" treated with sulfadiazine and sulfamerazine did not react to the pullorum agglutination test at 9 months of age. Mullen (1946) reported a difference of 14% in mortality with turkeys treated wifh 0.5% sulfamerazine in comparison to untreated controls in eleven flocks totaling 22,965 turkey poults. The results of these investigators showed that those sulfonamides which produced a relatively high blood level without toxicity gave promise of being effective chemotherapeutic agents for the treatment and control of pullorum disease. Three experiments were conducted to study the effect of sulfadiazine, sulfamerazine and sulfamethazine in the control of pullorum. Sulfamethazine was included because it gave blood levels equivalent to sulfamerazine at similar dosages and preliminary in vitro studies showed it to be effective against some gram negative organisms.
U. S. Pullorum-Clean flock were used in all experiments. The chicks were artificially infected, orally, with a broth culture of S. pullorum when they were transferred from the chick boxes to battery brooders. Feed containing the sulfonamides was placed in the feed troughs before the chicks were placed in the battery so that they had access to it immediately following inoculation. The sulfa drugs were administered in the feed for 5 days in experiment 1, for 10 days in experiment 2 and for 5 days in experiment 3. The chicks in all three experiments were weighed at 21 days of age and the average weight calculated. All chicks that died were examined bacteriologically for the presence of S. pullorum. The chicks in experiments 1 and 2 were inoculated with 500 million viable 5. pullorum organisms, while in experiment 3 the dose was reduced to approximately 5 million viable organisms per chick. In experiments 1 and 2 the amount of feed consumed during medication was determined and the average amount of drug intake per chick per day was computed. In experiment 1 the blood levels of each drug in various concentrations in the feed were secured at 24 hours, 48 hours and 96 hours after medication was started.
PROCEDURE Day old New Hampshire chicks from a 335
EXPERIMENT 1
Sulfadiazine was used in higher levels than sulfamerazine and sulfamethazine because Severns, Ripberts and' Card
Downloaded from http://ps.oxfordjournals.org/ at Uniwersytet Warszawski Biblioteka Uniwersytecka on May 12, 2015
(Received for publication December 7, 1946)
336
C. A. BOTTORFF AND J . S. KlSER TABLE 1.—Mortality and average weight pullorum disease experiment 1 No. chicks started
Sulfamethazine
0.75 0.50 0.25
Sulfadiazine
Sulfamerazine
Mortality 3 weeks
Average wt. in grams
No.
Percent
50 50 50
15 26 31
30 52 62
121 120 119
1.50 1.0 0.75
50 50 50
14 25 22
28 50 44
128 132 127
0.75 0.50 0.25
50 50 50
26 32 32
52 64 64
119 125 124
Infected, Untreated
—
50
47
94
84
Uninfected, Untreated
—
25
0
0
135
(1945) found that 2.0% sulfadiazine was required to obtain the same result as 0.5% of sulfamerazine. The drug was fed for 5 days. The highest mortality rate occurred during the first ten days in the infected controls. The mortality in the treated groups was negligible during the treatment period. When the drug was withdrawn from the diet, the mortality rate began to increase rapidly after five days, leveling off between the 17th to 19th day of age. Sulfamethazine showed a lower percentage of loss though there were not sufficient birds to show a significant difference. Sulfadiazine at 0.75% level gave as good protection as did sulfa-
merazine and sulfamethazine at the same levels, indicating that the three drugs might be of equal value in the treatment of pullorum disease. There was little difference in the average weights of the treated birds at 21 days of age. For mortality and average weights, see Table 1. Blood levels were obtained on a representative number of chicks from each group of treated birds. Both the free and total amounts of the drug were determined. The results of these tests are shown in Table 2. The amount of the conjugated drug was calculated by subtracting the percent of free drug from the percent of
TABLE 2.—Blood levels in milligrams percent pullorum disease experiment 1 24 hours Drug
% drug in mash
Free
Conjugated
48 hours Free
96 hours
Conjugated
Free
Conjugated
Sulfamethazine
0.75 0.50 0.25
19.13 12.90 8.90
1.33 0.00 0.00
19.13 . 15.57 9.34
2.22 0.00 0.00
28.47 16.90 11.12
0.44 0.00 0.00
Sulfamerazine
0.75 0.50 0.25
16.90 • 18.16 10.98
0.42 0.00 0.85
22.81 18.16 10.14
0.00 0.00 0.00
31.68 24.08 16.05
4.32 1.26 0.00
Sulfadiazine
1.50 1.00 0.75
28.00 24.80 27.20
1.60 3.20 1.60
40.40 30.40 23.20
8.00 10.80 8.00
39.20 31.20 25.60
8.00 7.60 6.20
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Percent drug in mash
Drug
337
U S E OF SULFONAMIDES IN CONTROL OF PULLORUM DISEASE
trols was 86% during the first 10 days, but it was 6% to 16% in the groups treated with high sulfa drug levels and 14% to 32% in those groups treated with low sulfa drug levels. In three to five days after the drug was withdrawn from the diet there was an increase in mortality that levelled off by the 20th to 21st day
TABLE 3.—Mortality and average weight pullorum disease experiment no. 2 Percent drug in mash
No. chicks started
Sulfamethazine
0.75 0.50 0.25
Sulfadiazine
Sulfamerazine
Mortality 3 weeks -No.
Percent
Average wt. in grams at 21 days
50 50 50
21 21 31
42 42 62
111 112 117
0.75 0.50 0.25
50 50 50
21 23 26
42 46 56
122 126 113
0.75 0.50 0.25
50 50 50
10 21 31
20 42 62
119 119 122
Infected, Untreated
—
50
47
94
93
Uninfected, Untreated
—
29
1*
2
177
Uninfected, Sulfamethazine
0.75
28
0
0
145
Uninfected, Sulfadiazine
0.75
28
0
0
176
Uninfected, Sulfamerazine
0.75
28
0
0
159
Drug
* Chick showed unhealed navel. 5. pullorum not isolated. EXPERIMENT 2
\
Due to the high mortality of the treated birds in experiment 1, it was decided to extend the treatment from 5 days to 10 days in experiment 2. Sulfadiazine was used at the same level as sulfamerazine and sulfamethazine to secure comparative values of the three drugs. In addition, to detect possible toxicity, three uninfected control groups were given sulfamethazine, sulfadiazine and sulfamerazine at a level of 0.75% in the feed for 10 days. The mortality rate in the infected con-
There was little difference in mortality at comparative levels in experiments 1 and 2. There was no significant difference in weight gains between any two groups of the treated infected birds. The treated infected birds made better weight gains than the infected untreated birds. In the treated uninfected groups the sulfadiazine-treated group made weight gains equivalent to the uninfected untreated controls. The sulfamethazine and sulfamerazine-treated groups made better weight gains than the infected treated
Downloaded from http://ps.oxfordjournals.org/ at Uniwersytet Warszawski Biblioteka Uniwersytecka on May 12, 2015
the total drug. There was little or no conjugation of sulfamethazine and sulfamerazine, but at high levels sulfadiazine showed some conjugation. Presumably the conjugated drug is inactive. This indicated that lower levels of sulfadiazine might give better protection than was secured at a 0.75% level.
338
C. A. BOTTORFF AND J. S. KlSEK
TABLE ABLE 4.—Average 4
amount drug consumed per chick in the controls, it was decided to reduce per day pullorum disease experiments 1 and 2 Percent of drug in mash
Drug
Drug Used Mg./Ch ick/Day Experi- Experiment 1 ment 2
0.75 0.50 0.25
44 28 14
57 35 17
Sulfadiazine
1.50 1.00 0.75 0.50 0.25
81 58 45
•—
— —
48 35 18
0.75 0.50 0.25
40 26 14
41 35 16
Sulfamerazine
groups but less than the uninfected untreated controls. The mortality and average weights for experiment 2 are given in Table 3. In experiments 1 and 2 the amount of feed consumed during medication was determined and the average amount of drug consumed per chick per day was computed. The results are given in Table 4. EXPERIMENT 3
Because of the extremely high mortality
TABLE 5.—Mortality and average weight pullorum disease experiment 3 Mortality 3 weeks No.
Percent
Average wt. in grams at 21 days
104 105 105
13 16 15
12.4 15.2 14.3
153 143 148
0.75 0.50 0.25
105 105 105
9 10 18
8.6 9.5 17.1
149 150 142
0.75 0.50 0.25
104 105 105
9 8 13
8.6 7.6 12.4
145 152 158
Infected, Untreated
—
105
67
64.0
119
Uninfected, Untreated
—
74
1.4
188
Percent drug in mash
No. chicks started
Sulfamethazine
0.75 0.50 0.25
Sulfadiazine
Sulfamerazine
Drug
* Chick showed unhealed navel. S. pullorum not isolated.
1*
Downloaded from http://ps.oxfordjournals.org/ at Uniwersytet Warszawski Biblioteka Uniwersytecka on May 12, 2015
Sulfamethazine
the infective dose in an effort to approach more nearly the average mortality that occurs in natural field outbreaks. This was accomplished by reducing the infective dose from 500 million to 5 million viable S. 'pullorum organisms per chick. Since no benefit was secured by the longer period of treatment in experiment 2, it was decided to treat the chicks for only 5 days. The number of chicks per group was increased to 105 chicks to try to reduce any errors that might occur where smaller lots of chicks were used. The mortality in the treated birds ranged from 8% to 12% at the high sulfa drug levels and from 12% to 17% at the low sulfa drug levels as compared to 64% in the untreated controls. Here again the mortality was checked during the period of treatment and the majority of deaths occurred between the 9th and 18th days. There was no significant difference in the weight gains of any of the treated groups. The mortality and average weights are given in Table 5. When the survivors of this group were blood tested at 12 weeks of age by the
USE OF SULFONAMIDES IN CONTROL OF PULLORUM DISEASE
rapid whole blood test for pullorum disease, approximately 90% of the birds showed a positive reaction to the test. DISCUSSION
No additional benefits appeared to be secured by feeding the drugs for more than five d a y s a t any one time. The infection was checked within five days if there was adequate consumption of the sulfonamide feed mixture.
In the dosages employed no toxic effects were observed or was there any significant effect on the weight gains of birds treated by any of the three drugs. In all experiments there was a minimum of 30% to a maximum of 56% reduction in mortality. SUMMARY 1. Sulfadiazine, sulfamethazine and sulfamerazine were equally effective in the reduction of mortality from pullorum disease in baby chicks. 2. A minimum of 30% to a maximum of 56% reduction in mortality occurred in treated groups as compared to untreated controls. 3. No significant difference in weight gains was found after 21 days in the treated groups of birds. 4. No evidence of toxicity was noted. 5. Ninety percent of surviving chicks reacted to the rapid whole blood test for pullorum disease at 12 weeks of age. REFERENCES
Mullen, F. E., 1946. Sulfamerazine as a prophylactic in pullorum disease in poults. Jour. Am. Vet. Med. Assoc. 108: 163-164. Severns, J. M., E. Roberts, and L. E. Card, 1945. The effect of sulfonamides in reducing mortality from pullorum disease. Poultry Sci. 24: 155-158.
Downloaded from http://ps.oxfordjournals.org/ at Uniwersytet Warszawski Biblioteka Uniwersytecka on May 12, 2015
There was no significant difference between sulfadiazine, sulfamethazine and sulfamerazine in their effectiveness in the reduction of mortality under the conditions of these experiments. The first two experiments failed to give as good results as experiment 3 due to the overwhelming infective dose of S. pullorum organisms. When the infective dose per chick was reduced so that it would more nearly approximate the degree of infection that occurs in a severe natural outbreak, better protection was secured. It is possible that this mortality could be reduced further by using two or three treatments of three or four days duration with four to five days between treatments. This time interval is based on the recurrence of mortality beginning five days after withdrawal of the drugs from the diet in all three experiments.
339