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Eimeria tenella: Specific reversal of t-butylaminoethanol toxicity for parasite and host by choline and dimethylaminoethanol
EXPERIMENTAL PARASITOLOGY 47, 13-23
(19%)
Eimeria fenellu: Specific Reversal of t-Butylaminoethanoi Toxicity Parasite and Host by Choline and Dimethylaminoethanol
for
E. C. MCMANUS, E. F. ROGERS, B. M. MILLER, F. R. JUDITH, K. D. SCHLEIM, AND G. OLSON Merck Sharp 6 Dohme Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065, U.S.A. (Accepted
for publication
17 October
1978)
MCMANUS, E. C., ROGERS, E. F., MILLER, B. M., JUDITH, F. R., SCHLEIM, K. D., AND OLSON, G. 1979. Eimeria tenella: Specific reversal of t-butylaminoethanol toxicity for Experimental Parasitology 47, parasite and host by choline and dimethylaminoethanol. 13-23. t-Butylaminoethanol is an anticoccidial compound that is related structurally and choline. Toxic effects to the metabolically active substances, dimethylaminoethanol, of t-butylaminoethanol for chickens and Eimeria tenella are specifically overcome by feeding sufficient amounts of dimethylaminoethanol or choline. Dietary concentrations of the two above metabolites required to totally overcome toxic effiects of t-butylaminoethanol were determined and are expressed as the reversal ratio, inhibitor (t-butylaminoethanol) :metabolite. The inhibitor:choline ratio for total reversal of toxic effects of the inhibitor in chickens is approximately 1:lO over a concentration range of inhibitor from 0.019 to 0.05%. The inhibitor:choline ratio for reversal of antiparasitic effects is approximately 1:200 with a concentration of 0.01% inhibitor. The inhibitor:dimethylaminoethanol ratio for reversal of toxic effects of the inhibitor in the chicken is approximately 1: 7 with a concentration of 0.015% inhibitor. The inhibitor: dimethylaminoethanol ratio for reversal of antiparasitic effects is approxmately 1:20 wth a concentration of 0.01% inhibitor. INDEX DESCRIPTORS: Protozoa, parasitic; Eimeria tenella; Chicken; Chemotherapy; Synergism; Metabolite inhibitor; t-butylaminoethanol; Methylaminoethanol; Dimethylaminoethanol; Choline; Sulfaquinoxaline; Pyrimethamine.
of experiments with t-butylaminoethanol concerned with anticoccidial effects, growth depressing effects for young chickens and their reversibility by choline and by dimethylaminoethanol.
INTRODUCTION
When the anticoccidial activity of t-butylaminoethanol for Eimeria tenetla was discovered in our laboratories, the structural relationship to choline and precursors of choline was apparent and reversal experiments were conducted. Also, combinations of sulfaquinoxaline and t-butylaminoethanol were tested, and a highly significant synergistic interaction was detected. Preliminary results have been reported (McManus and Rogers 1969; McManus et al. 1975). This paper contains details
MATERIALS
Anticoccidial
AND METHODS
Eficacy
Two highly pathogenic strains of Eim&a tenella were used for the experiments. For the initial work a strain, designated “Laboratory Strain-l&” which has 13 0014-4894/79/010013-11$02.00/0 Copyright @ 1979 by Academic Press, Inc. Al1 rights of repI-oduction in any form resewed.
14
MCMANUS
been maintained in the laboratory for more than 20 years was used. For later experiments in which t-butylaminoethanol was combined with sulfaquinoxaline and pyrimethamine, a strain isolated in 1971 from a field outbreak in Japan, designated “DP-447,” was used. Two-week-old-sex-balanced white cross chickens in groups of 10 were weighed, placed in batteries, and fed a standard laboratory ration in which test chemicals were blended. Noninfected, nonmedicated and infected, nonmedicated groups were fed the basal ration. One day after the commencement of the test, all but noninfected, nonmedicated groups were inoculated with 50,000 sporulated oocysts per bird of E. ten&a. Eight days after inoculation, chickens were weighed, sacrificed, and ceca were assigned coccidial gross lesion scores from 0 to 4. Cecal lesion scoring was similar to that employed by Johnson and Reid (1970). The ceca were homogenized in water, and aliquots were taken for oocyst counts. Parameters used to evaluate efficacy of the compound were mortality rate, growth, severity of gross coccidial lesions, and numbers of oocysts produced. For each group of birds, the several parameters were summarized into one number by the use of the “anticoccidial index” (Anonymous 1960; McManus et ~2. 1968). A brief description of the derivation of the index follows: The anticoccidial index is the sum of (1) the percentage survival plus (2) the percentage relative weight gain minus (3) the lesion index, and minus (4) the oocyst index. Each of the contributing components of the anticoccidial index was computed as follows: (1) The percentage survival (O100) contributes the corresponding number of points (o-100). (2) The percentage relative weight gain, calculated by dividing the weight gain of the group by the average weight gain of the noninfected, nonmedicated Ygrouns, and multinlied bv, A. L
ET
AL.
100 (to convert to percentage) contributes 0 to 100 points or more or less. (3) The observed average lesion score (04.0) is multiplied by 10 to become the corresponding lesion index (040). (4) The oocyst count of a test group is divided by the mean oocyst count of the infected, nonmedicated groups, then multiplied by 100 (to obtain an observed percentage of contro1). This number is muhiphed by 0.4 to become the assigned oocyst index. The oocyst index is 0 to a maximum of 40 except in the case of the infected, nonmedicated control groups. In this latter instance the index for in&vi&al infected, nonmedicated groups may be more or less than 40 but the mean index for these groups is always 40. Therefore survival and weight gain are each assigned equal importance and gross lesions and oocyst counts are assigned equal importance. Weight gains and survival each contribute 2.5 times the numerical value of the gross lesions and oocyst counts to the anticoccidial index. An anticoccidial index reresponse of 180 or higher for a medication is rated good. Two experimental designs were used to test for reversibility of anticoccidial activity of t-butylaminoethanol by choline. In the first and simpler design, 0.05% t-butylaminethanol (the minimal effective level) was tested alone and combined with a range of levels of choline. In the second experimental design, the level of t-butylaminoethanol was decreased by the use of compounds that are synergistic with it. As stated above, t-butylaminoethanol is synergistic with sulfaquinoxaline against E. tenellu. Sulfaquinoxaline is synergistic with pyrimethamine (Lux 1954; KendaII and Joyner 1956). The same two designs were also used for efficacy reversal experiments with dimethylaminoethanol. Tolerance For evaluation of toxicity of the test compounds in young chickens, battery tol-
Eimeria
tenella:
TABLE Efficacy of t-Butylaminoethanol Dietary LButylaminoethanol HCl
Methylaminoethanol HCl
(%)
(%)
N0lle
NOIE (Noninfected,
NOIX
NOW (Infected.
against Eimeriu
I
tenek Laboratory Strain-18, and Efficacy Reversal Parameter
No. birds
medication
Mortality
Choline chloride
Dimethylaminoethanol HCl
15
CHOLINE REVERSAL
(%)
(%)
Relative weight gain
Average lesion score
(%)
(%) NOIE nonmedicated NOlIt nonmedicated NOLIe NOIE NOIE
Basal* controls) Basal controls) Basal Basal Basal
Average No. oocysts/ bird x 10’
Anticoccidid index
40
0
100
0.0
0.0
200
40
54
54
3.8
5.6
22
20 20 20
0 0 0
60 80 86
3.8 2.2 0.2
6.4 2.8 0.0
82 138 184
20 20 10
0 0 0
94 98 91
0.2 1.1 0.8
0.5 3.4 1.8
188 163 170
0.0125 0.025 0.05
NOW NOlIe NOnI?
0.05 0.05 0.05
NOIll NOIE NOIE
NOW. NOM NOW3
Basal Basal Basal
0.05 0.05 0.05
NOW NIXE NOIE!
0.025 0.05 0.1
BSSSl Basal Basal
20 20 10
0 0 0
88 91 90
0.5 1.6 3.2
0.7 1.8 10.6
178 162 118
0.05
0.05
NOlIt?
BE&Sal
10
0
89
0.0
0.1
188
a Basal
diet
contained
0.267%
choline
+ 0.5 + 1.0 + 2.0
Cl.
erance tests in noninfected birds were performed which continued from 4 to 22 days of age. In these experiments, dayold white cross birds were placed in electrically heated wire-floored batteries in a temperature-controlled room and fed the basal nonmedicated ration for 3 days. At 4 days of age the chickens were weighed and sorted into groups with 2-g weight increments, They were randomly assigned to cages containing four to eight birds with similar weight distributions and total weights. Group weights were taken at 6 and 12 days on test, and individual weights were taken at the termination of the experiment on test Day 18. The same basal diet was used throughout, with the exception of one tolerance experiment. The diet for the one tolerance experiment contained 0.188% choline chloride instead of 0.267% choline chloride. Choline analyses were conducted by the Wisconsin Alumni Research Foundation, Madison, Wisconsin. Choline bitartrate was used for dietary supplementation to the choline chloride already present in the basal diet because it is one of the least hygroscopic salts of choline. Weights of supple-
mental choline bitartrate were corrected to give molar choline chloride equivalents and all choline dietary concentrations appearing in the text, tables, and figures indicate the chloride salt. All medicated diets in Tables II and III contained 0.0005% ethopabate. Hydrochloride salts of t-butylaminoethanol and dimethylaminoethanol were used in all experiments and dietary concentrations appearing herein are of the HCI salts. RESULTS
Anticoccidial
Eficacy
A dietary level of 0.05% t-butylaminoethanol showed good activity against a virulent infection of Eimeriu tenella (Table I). Choline, 1.0 and 2.0%, was added to the basal diet, which already contained 0.267% choline, for the choline reversal experiments summarized in Table I. A total of 1.267 to 2.267% choline incompletely reversed the efficacy of t-butylaminoethanol. The choline which is supplemented and the 0.267% which is already in the basal diet will be added together in the presen-
16
MCMANUS TABLE Efficacy
of Synergistic Dietary
ET AL. II
against Eimeria tenella DP-447
Combinations
and Reversal
by Cldinc -
raramcter
medication birds
Sulfaquinoxalino
t-Butylaminoethanol HCl
(%)
Pyrimethamine
Choline chloride
iUortality
(%I
(%)
Relative weight gain
Average lesion score
(‘%I)
(%I
(%) NlXle (Noninfected, NOIE NOWZ (Infected,
NCJIle
NOlli2 nonn~edicated None nonmedicated
Average NO. ooeysts/ bird X IO”
Anticoccidisl index
Basal* controls) Basal controls)
20
0
100
0.0
0.0
200
50
30
G3
4.0
37.3
53
N0ne None
O.OOG 0.006
0.0003 0.0005
Basal Basal
10 10
30 10
83 8X
2.7 2.6
34.9 29.2
91 121
0.01 0.015 0.02
0.006 0.006 0.006
0.0003 0.0003 0.0003
&Ma1 Basal Basal
10 10 10
0 0 0
95 87 94
2.5 1.4 0.4
12.0 6.9 1.1
157 lG7 189
0.01 0.015 0.02
0.006 0.006 0.006
0.0005 0.0005 0.0005
10 10 10
0 0 0
94 9G 87
0.X 0.3 0.4
I.5 1.9 2.5
184 191 180
0.01 0.015 0.02
0.006 O.OOB 0.006
0.0003 0.0003 0.000~
Basal Basal Basal
+ 0.5 + 0.5 + 0.5
10 10 10
0 0 0
09 91 97
1.8 1.9 0.8
8.1 0.5 7.0
172 16s 181
0.01 0.015 0.02
0.006 0.006 0.006
0.0005 0.0005 0.0005
Rassl Basal Basal
+ 0.5 + 0.5 + 0.5
10 10 10
0 0 0
98 98 89
0.9 0.0 0.2
1.4 0.0 0.0
IS7 198 187
0.01 0.015 0.02
0.006 0.006 O.OOG
0.0003 0.0003 0.0003
Ram1 Basal nasal
+ + +
1.0 1.0 1.0
10 10 10
0 0 0
94 92 94
2.1 1.1 1.2
1G.S 4.0 4.9
1x 177 177
0.01 0.015 0.02
0.006 0.006 0.006
0.0005 0.0005 0.0005
Bass1 Basal Basal
+ + +
1.0 1.0 1.0
10 10 10
0 0 0
92 93 97
1.5 I.4 0.5
15.2 7.8 1.3
161 171 191
0.01 0.015 0.02
0.006 0.006 0.006
0.0003 0.0003 0.0003
Basal Basal Basal
+ 2.0 + 2.0 + 2.0
10 10 10
20 0 0
80 GO 8ti
3.5 3.1 1.2
2’S.0 32.2 8.7
95 103 lfi.5
0.01 0.015 0.02
0.006 0.006 0.006
0.0003 0.0005 0.0005
Basal Basal Basal
+ 2.0 + 2.0 + 2.0
10 10 10
10 0 0
80 7s 88
2.!) 1.9 1.1
11.8 9.2 12.5
134 149 164
0 Basal
diet
contained
0.267%
choline
Cl.
tation. The levels appearing in the text, tables, and figures are the total dietary concentration. In the second efficacy protocol (Materials and Methods) synergistic combinations were used. t-Butylaminoethanol is synergistic with sulfaquinoxaline against E. tenella. Sulfaquinoxaline is synergistic with pyrimethamine. A mixture of 0.006% sulfaquinoxaline, 0.0003 to 0.00057, pyrimethamine, and 0.01 to 0.02% t-butylaminoethanol was highly effective in chickens given heavy exposure to E. tenella (Tables II and III).
Choline reversal experiments were conducted with sulfaquinoxaline and pyrimethamine combined with a range of levels of t-butylaminoethanol from 0 to 0.02% (Table II). Choline, 2.267%, totally reversed the anticoccidial effect contributed by 0.01% t-butylaminoethanol in synergistic combination with 0.0067, sulfaquinoxaline and 0.0003 to 0.00057, pyrimethamine. Based on all parameters of the experiment, the reversing effect of 2.267% choline was such that there was little difference in response to 0.006% sulfaquinoxaline plus pyrimethamine with or without 0.01% t-butylaminoethanol.
Eimeria tenda:
CHOLINE
TABLE
III
Efficacy of Synergistic Combinations against Eimeria and Reversal by Dimethylaminoethanol Dietary t-Butylaminoethanol HCI
Sulfaquinoxaline (%)
medication Pyrimethamine (%)
tenella DP-447 Parameter
No. hirds Dimethylsminoethanol HCl
17
REVERSAL
MOP tality (%)
Relative weight gain
Average lesion score
(%)
(%)
(%) NOIE
NOI%2
(Noninfected, NOW
(Infected,
NOIll?
nonmedicated NOIll? NOW nonmedicated
Average NO. oocysts/ bird x 10”
Anticoccidial index
NOW
20
0
100
0.0
NOW
50
38
68
4.0
27.6
50
NOW
0 0 0
103 99 97
1.1 0.5 0.0
3.0 3.7 0.0
188 189 197
controls) controls)
0.01 0.015 0.02
0.006 0.006 0.006
0.0003 0.0003 0.0003
NOW
10 10 10
0.01 0.015 0.02
0.006 0.006 0.006
0.0005 0.0005 0.0005
None NOW None
10 10 10
0 0 0
98 97 106
0.8 0.0 0.0
3.7 0.0 0.0
185 197 206
0.01 0.015 0.02
0.006 0.006 0.006
0.0003 0.0003 0.0003
0.05 0.05 0.05
10 10 10
0 0 0
9.5 107 101
1.2 0.8 0.4
11.8 3.8 0.8
166 193 196
0.01 0.015 0.02
0.006 0.006 0.006
0.0005 0.0005 0.0005
0.05 0.05 0.05
10 10 10
0 0 0
104 99 103
0.7 0.2 0.6
2.9 0.8 4.0
193 196 191
0.01 0.015 0.02
0.006 0.006 0.006
0.0003 0.0003 0.0003
0.1 0.1 0.1
10 10 10
10 0 0
95 96 82
2.5 3.1 1.5
12.0 36.1 6.2
143 125 158
0.01 0.015 0.02
0.006 0.006 0.006
0.0005 0.0005 0.0005
0.1 0.1 0.1
10 10 10
0 0 0
104 90 101
1.2 1.8 0.2
7.6 3.3 1.0
181 167 198
0.01 0.015 0.02
0.006 0.006 0.006
0.0003 0.0003 0.0003
0.2 0.2 0.2
10 10 10
0 10 10
68 85 86
3.2 3.1 3.3
13.7 22.2 25.2
116 112 106
0.01 0.015 0.02
0.006 0.006 0.006
0.0005 0.0005 0.0005
0.2 0.2 0.2
10 10 10
10 0 0
96 99 87
2.4 2.2 2.0
23.1 12.8 11.2
129 158 151
NOIE
Dimethylaminoethanol, O.l%, brought about a substantial but incomplete reversal of the anticoccidial action of 0.05% t-butylaminoethanol (Table I), Methylaminoethanol had no effect in reversal experiments with t-butylaminoethanol (Table I). Dimethylaminoethanol, 0.27~~ totally reversed the anticoccidial activity contributed by 0.01% t-butylaminoethanol in the synergistic combination with 0.006% sulfaquinoxaline and 0.0003 to 0.0005% Dimethylaminoethanol, pyrimethamine. 0.2%, sometimes but not consistently reversed 0.015 to 0.02% t-butylaminoethanol in the synergistic combination. Levels
below 0.2% had less reversing effect (Table III). Noninfected, medicated control groups of birds were tested with the highest dietary levels of the mixtures of chemicals used in the reversal experiments of Tables II and III. These tests were toxicity tests which followed the protocol of the efficacy assays. They are not to be confused with the more precise tolerance tests in chicks which ran from 4 to 22 days of age. The tests with noninfected, medicated controls were necessary to assure that toxicity of the several chemicals was not affecting the efficacy results. The mixtures
1s
MCMANUS
Sulfaquinoxaline (70)
Choline chloride (5%)
t-Butyl aminoethanol HCl (%I
Pyrimethamine cm
None None None None None
None None None None None
None None None None None
Basala Basal Basal Basal Basal
0.01
0.0003
0.006
0.01
0.0003
0.006
0.015 0.015 0.02 0.02
0.0003 0.0003 0.0003 0.0003
0.006 0.006 0.006 0.006
Basal Basal Basal Basal Basal Basal
0.02 0.02 0.02 0.02
0.0003 0.0003 0.0003 0.0003
0.006 0.006 0.006 0.006
Basal Basal Basal Basal
= Basal diet contained
ET
AL.
Dimethylaminoethanol HCl (75)
Wright gain Cd
270 278 271 267 285 + + + + + +
2.0 2.0 2.0 2.0 2.0 2.0
279 256 2.58 267 259 270 282 280 279 271
0.2677{; choline Cl.
tested did not significantly depress weight gain (P Q 0.05, Table IV). Tolerance Eighteen-day tolerance experiments demonstrated marked toxicity with high mortality for 0.04 and O.OScjo t-butylaminoethanol. Toxicity was not well defined in the shorter (g-day) efficacy tests which ran from 14 to 23 days of age. Eighteen-day tolerance tests were performed in one experiment with a commercial diet containing 0.188% choline. Weight gain is plotted against graded concentrations of t-butylaminoethanol (Fig. 1). The dose-response curve intercepted zero growth depression at 0.019% t-butylaminoethanol. Details of the experiment are presented in the legend of Fig. 1. A relationship of choline to tolerance by the chicken of t-butylaminoethanol re-
mained to be established. In further tolerance experiments, diets were supplemented with a fixed toxic level of t-butylaminoethanol, and choline was made the variable. Weight gain is plotted against (Figs. 2 graded choline concentrations and 3). In tests with 0.04% t-butylaminoethanol, the intercept for zero growth depression is 0.451% choline (Fig. 2). In tests with diets supplemented with 0.05% t-butylamilloethanol, the intercept is 0.513c/o choline (Fig, 3). Mortality due to t-butylaminoethanol was prevented by choline (Figs. 2 and 3). Details of the experiments are presented in the legends of the figures. Similary designed toxicity-reversal exdimethylaminoethanol periments with clearly demonstrated that it, as well as choline, was capable of reversing the adverse effects of t-butyaminoethanol on growth and survival of young chickens. The toxicity of 0.04 and 0.05% t-butyl-
Eimeria
tenella:
19
CHOLINE REVERSAL
I IO-
100 -
. .
50 IMortality:
4%
0%
4%
I 0.01
I
I
I
0.02
0.03
0.04
Percent
Dietary
t-Butylaminoethanol
2 I%
HCl
FIG. 1. Effect of feeding graded levels of t-butylaminoethanol upon the growth of chickens from 0.5 to 3 weeks of age. Each dot represents the relative percentage weight gain of one pen of four chickens. The horizontal lines represent the average weight gain (solid line) and the 0.95 probability confidence interval (dashed lines) of 20 pens of nonmedicated control chickens ( 100% = 362.1 g f 3.7%). The sloping lines are the dose-response curve (solid line) and its 0.95 probability confidence interval (dashed lines) for the growth response of 18 pens of chickens fed 0.02 to 0.04% t-butylaminoethanol HCl. The equation for the doseresponse curve is y = -52.99 - 88.96x where x = log percentage dietary t-butylaminoethanol HCl and y = relative percentage weight gain. In the calculation of linear regression, the responses were weighted to reflect mortality in some of the pens.
aminoethanol was incompletely reversed by 0.1% dimethylaminoethanol, the highes2 level tested. Mortality was totally prevented and weight gain was markedly improved but not fully returned to norma1 (Table V). DISCUSSION
The anticoccidial efficacy and the adverse effects on growth and survival of young chickens of t-butylaminoethanol are reversible by choline and by dimethylaminoethanol. The ratios of the dose of inhibitor to metabolite for reversal can be approximated. Unusually large quantities of choline were required for anticoccidial reversal of
t-butylaminethanol. A dietary level of 0.05% t-butylaminoethanol was incompletely reversed by 2.267% choline (Table I) giving a reversal ratio of 1 part inhibitor to > 45 parts metabolite. Although the 2.267% level of choline was not toxic, it was probably close to a growth depressing dietary concentration (Melass et al. 1946), and efficacy reversal experiments with dietary levels of choline higher than 2.267% were not attempted. Synergistic mixtures of t-butylaminoethano1 with suIfquinoxaline and pyrimethamine permitted lower medication levels of t-butylaminoethanol (Table II). In the synergistic combination, the anticoccidial contribution of 0.010/o t-butylaminoethanol was reversed by
20
MCMANUS
ET
AL.
t 100
t
60
0.267
0.367 Percent
Dietory
0.467
Of i67
Choline Cl
FIG. 2. Effect of graded levels of dietary choline Cl fed in combination with 0.04% t-butylaminoethanol HCl upon the growth of chickens from 0.5 to 3 weeks of age. Each dot represents the relative percentage weight gain of one pen of eight chickens. The horizontal line represents the average weight gain (solid line) and its 0.95 probability confidence interval (dashed lines) of 12 pens of nonmedicated control chickens ( 100% = 292.6 g 2 4.9%). The sloping lines are the dose-response curve (solid line) and its 0.95 probability confidence interval (dashed lines) for the growth response of 17 pens of chickens fed 0.267 to 0.467% choline Cl in combination with 0.04% t-butylaminoethanol HCl. The equation for the curve is y = 132.69 + 94.42x where x = log percentage dietary choline Cl and y = relative percentage weight gain. In the calculation of linear regression, the responses were weighted to reflect mortality in scme of the pens.
2.267% choline, giving a reversal ratio of about 1 part inhibitor to 200 parts metabolite. The anticoccidial activity of O.OSY, t-butylaminoethanol was substantially but incompletely reversed by 0.1% dimethylaminoethanol (Table I) indicating a reversal ratio of 1 part inhibitor to > 2 parts metabolite. In further antiparasitic reversal experiments with dimethylaminoethanol employing the synergistic mixture of t-butylaminoethanol, sulfaquinoxaline, and pyrimethamine (Table III), the anticoccidial contribution of 0.01% t-butylaminoethanol was reversed by 0.2% dimethylaminoethanol which indicates a
reversal ratio of approximately 1 part inhibitor to 20 parts metabolite. For calculation of the above ratio, the contribution of the choline in the basal diet to reversal of the anticoccidial action by dimethylaminoethanol is relatively unimportant because the anticoccidial reversal ratio of t-butylaminoethanol to choline is unusually low. The toxicity for the chicken of 1 part t-butylaminoethanol was overcome by approximately 10 parts dietary choline. Levels of 0.0191, 0.01, and O.OSY, t-butylaminoethanol were tolerated by the young chicken when choline levels were, respectively, 0.188, 0.451, and 0.513r/o. The lin-
Eimeriu
tenella:
110
CHOLINE I
I -s-m
21
REVERSAL I
l
2%
0%
l l
l
100
l
60 Mortolity:
15%
50% I
I
I
I
0.267
0.367
0.467
0.66 7
Percent
Dietory
Choline Cl
FIG. 3. Effect of graded levels of dietary choline Cl fed in combination with 0.05% t-butylaminethanol HCl upon the growth of chickens from 0.5 to 3 weeks of age. Each dot represents the relative percentage weight gain of one pen of eight chickens. The horizontal line represents the average weight gain (solid line) and its 0.95 probability confidence interval (dashed lines) of 12 pens of nonmedicated control chickens ( 100% = 292.6 g f 4.9%). The sloping lines are the dose-response curve (solid line) and its 0.95 probability confidence interval (dashed lines) for the growth response of 18 pens of chickens fed 0.267 to 0.467% choline Cl in combination with 6.05% t-butylaminoethanol HCl. The equation for the doseresponse curve is y = 118.21+ 62.81~ where x = log percentage dietary choline Cl and y = relative percentage weight gain. In the calculation of linear regression, the responses were weighted to reflect mortality in some of the pens.
ear relationship of the experimentally determined ratios of inhibitor to metabolite is presented graphically in Fig. 4. The correlation coefficient (T) for linearity is 0.99. Additional evidence for linearity is the fact that the extrapolated curve is close to intercepting x = 0, y = 0. This is expected. Toxicity for the chicken of 0.04 and 0.05% t-butylaminoethanol was almost totally reversed by 0.1% dimethylaminoethanol. Weight gain was markedly improved but not returned completely to normal. Mortality was totally prevented (Table V). For calculation of the toxicityreversal ratio of t-butylaminoethanol to dimethylaminoethanol, the contribution to
reversal by the choline in the basal diet (0.267%) is important. Figure 4 indicates that 0.267% choline reverses toxicity of approximately 0.0257, t-butylaminoethanol. In the basal diet supplemented with 0.04% t-butylaminoethanol, a 0.025% portion of the 0.04% inhibitor is reversed by the choline in the basal diet giving a difference of 0.0470 - 0.025% = 0.015%. For reversal of toxicity for the chicken, the inhibitor to dimethylaminoethanol ratio is approximately 0.015: 0.1 or approximately 1:7. A singular feature of the results presented is the large amount of dietary metabolite required for reversal of t-butylaminoethanol. The quantity of choline
22
MCMANUS
I
0.05 E
B
2 5 f Q % m A 6 .-t 0 E g a”
ET
I
AL.
I
I
I
0.04
0.03
0.02 t
0.01
I
0
0
0. 6 Percent
Dietary
Choline
Cl
FIG. 4. Tolerance of t-butylaminoethanol with increasing levels of choline. Each dot represents the concentration of choline required to overcome growth depressing effects in the chicken of a level of t-butylaminoethanol. The equation for the curve is y = 0.00163 -I0.09046x where x = percentage dietary choline Cl and y = percentage dietary t-butylaminoethanol HCI.
TABLE Reversal
of Toxicity
Weight
of t-Butylaminoethanol
gain, relative
percentage Dietary
Expt No.
None
A. Diets supplemented
V by Dirnethylaminoethanol
of nonmedicated
percentage
control
dimethylaminoethanol
0.025
with 0.04y0 t-butylaminoethanol
chickens HCI
0.05
0.1
HCl
1
87.0 (7/8)G 73.3 (6/S)
87.6 (S/S) 83.4 (7/8)
92.4 (8/S) 101.9 (8/8)
97.7 (8/S) 93.5 (8/8)
2
64.7 (6/S) 51.7 (2/8)
67.4 (7/8) 87.4 (7/S)
73.3 (7/8) 8.5.2 (S/S)
78.4 (8/8) 88.6 (8/8)
69.2
81.4
88.2
89.6
9
3
0
Average Mortality
(72)
34
B. Diets supplemented
with 0.05’s t-butylaminoethanol
HCl
1
77.0 (5/S) 70.3 (S/8)
85.8 (5/8) 100.5 (3/S)
101.5 (8/8) 87.8 (8/8)
90.4 (S/8) 86.6 (8/8)
2
54.4 (S/8) 40.7 (l/S)
70.3 (7/S) 87.1 (3/S)
77.3 (S/B) 92.3 (6/8)
80.7 (S/8) 87.0 (8/8)
60.6 62
85.9 44
89.7 12
86.2 0
Average Mortality (1Number
(70)
in parentheses
is number
of survivors
in each pen of eight birds.
Eimeria
tenekz: CHOLINE
required for reversal of the antiparasitic effect is especially large. Choline is a quaternary cation and therefore is totally ionized in solution. Dimethylaminoethanol is a tertiary amine (pK, 9.26). At pH 7.4, approximately is non1.4% of dimethylaminoethanol ionized. This difference between quaternary nitrogen compounds and tertiary amines has important implications for diffusion across biological membranes. These membranes generally are lipid in nature and nonionized compounds diffuse across such barriers at a more rapid rate than ionized compounds. Movement of choline across membranes of the parasite may be restricted because of its ionization properties. This could relate to the large amount of choline required by the parasite for reversal of the inhibitor. A much smaller amount of choline is required for reversal of toxicity of t-butylaminoethanol for the chicken than for the parasite. In other words there is a marked species specificity for the reversal by choline. Obviously, a favorable therapeutic ratio is achieved with a compound whose toxicity is more easily overcome for the host than for the parasite.
23
REVERSAL
REFEREN1960. Technical Bulletin, AMPROL, an effective new coccidiostat for broilers. Merck and Co., Inc., Rahway, New Jersey. JOHNSON, J., AND REID, W. M. 1970. Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments with chickens. Experimental Parasitology 28, 30-36. KENDALL, S. B., AND JOYNER, L. P. 1956. The potentiation of coccidiostatic drugs by pyrimethamine. VeterinuTy Record 68, 119-121. Lux, R. E. 1954. The chemotherapy of Eimeria tenella: I, Diaminopyrimidines and dihydroAntibiotics and Chemotherapy 4, triazines. 971-977. MCMANUS, E. C., CAMPBELL, W. C., AND CUCKLER, A. C. 1968. Development of resistance to quinoline coccidiostats under field and laboraJournal of Parasitology 54, tory conditions. 1190-1193. MCMANUS, E. C., AND ROGERS, E. F. 1969. Anticoccidial effect of t-butylaminoethanol, its reversibility by dimethylaminoethanol and its synergistic interaction with sulfonamides. Federation Proceedings 28, 390. MCMANUS, E. C., TAMAS, T., JUDITH, F. R., AND ROGERS, E. F. 1975. Anticoccidial and growth depressing effects in chicks of t-butylaminoethanol and reversibility by choline and precursors. Poultry Science 54, 1793. MELASS, V. H., PEARSON, P. B., AND SHERWOOD, R. M. 1946. Toxicity of choline in the diet of growing chickens. Proceedings of the Society of Experimental Biology and Medicine 62, 174176. ANONYMOUS.
(19%)
Eimeria fenellu: Specific Reversal of t-Butylaminoethanoi Toxicity Parasite and Host by Choline and Dimethylaminoethanol
for
E. C. MCMANUS, E. F. ROGERS, B. M. MILLER, F. R. JUDITH, K. D. SCHLEIM, AND G. OLSON Merck Sharp 6 Dohme Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065, U.S.A. (Accepted
for publication
17 October
1978)
MCMANUS, E. C., ROGERS, E. F., MILLER, B. M., JUDITH, F. R., SCHLEIM, K. D., AND OLSON, G. 1979. Eimeria tenella: Specific reversal of t-butylaminoethanol toxicity for Experimental Parasitology 47, parasite and host by choline and dimethylaminoethanol. 13-23. t-Butylaminoethanol is an anticoccidial compound that is related structurally and choline. Toxic effects to the metabolically active substances, dimethylaminoethanol, of t-butylaminoethanol for chickens and Eimeria tenella are specifically overcome by feeding sufficient amounts of dimethylaminoethanol or choline. Dietary concentrations of the two above metabolites required to totally overcome toxic effiects of t-butylaminoethanol were determined and are expressed as the reversal ratio, inhibitor (t-butylaminoethanol) :metabolite. The inhibitor:choline ratio for total reversal of toxic effects of the inhibitor in chickens is approximately 1:lO over a concentration range of inhibitor from 0.019 to 0.05%. The inhibitor:choline ratio for reversal of antiparasitic effects is approximately 1:200 with a concentration of 0.01% inhibitor. The inhibitor:dimethylaminoethanol ratio for reversal of toxic effects of the inhibitor in the chicken is approximately 1: 7 with a concentration of 0.015% inhibitor. The inhibitor: dimethylaminoethanol ratio for reversal of antiparasitic effects is approxmately 1:20 wth a concentration of 0.01% inhibitor. INDEX DESCRIPTORS: Protozoa, parasitic; Eimeria tenella; Chicken; Chemotherapy; Synergism; Metabolite inhibitor; t-butylaminoethanol; Methylaminoethanol; Dimethylaminoethanol; Choline; Sulfaquinoxaline; Pyrimethamine.
of experiments with t-butylaminoethanol concerned with anticoccidial effects, growth depressing effects for young chickens and their reversibility by choline and by dimethylaminoethanol.
INTRODUCTION
When the anticoccidial activity of t-butylaminoethanol for Eimeria tenetla was discovered in our laboratories, the structural relationship to choline and precursors of choline was apparent and reversal experiments were conducted. Also, combinations of sulfaquinoxaline and t-butylaminoethanol were tested, and a highly significant synergistic interaction was detected. Preliminary results have been reported (McManus and Rogers 1969; McManus et al. 1975). This paper contains details
MATERIALS
Anticoccidial
AND METHODS
Eficacy
Two highly pathogenic strains of Eim&a tenella were used for the experiments. For the initial work a strain, designated “Laboratory Strain-l&” which has 13 0014-4894/79/010013-11$02.00/0 Copyright @ 1979 by Academic Press, Inc. Al1 rights of repI-oduction in any form resewed.
14
MCMANUS
been maintained in the laboratory for more than 20 years was used. For later experiments in which t-butylaminoethanol was combined with sulfaquinoxaline and pyrimethamine, a strain isolated in 1971 from a field outbreak in Japan, designated “DP-447,” was used. Two-week-old-sex-balanced white cross chickens in groups of 10 were weighed, placed in batteries, and fed a standard laboratory ration in which test chemicals were blended. Noninfected, nonmedicated and infected, nonmedicated groups were fed the basal ration. One day after the commencement of the test, all but noninfected, nonmedicated groups were inoculated with 50,000 sporulated oocysts per bird of E. ten&a. Eight days after inoculation, chickens were weighed, sacrificed, and ceca were assigned coccidial gross lesion scores from 0 to 4. Cecal lesion scoring was similar to that employed by Johnson and Reid (1970). The ceca were homogenized in water, and aliquots were taken for oocyst counts. Parameters used to evaluate efficacy of the compound were mortality rate, growth, severity of gross coccidial lesions, and numbers of oocysts produced. For each group of birds, the several parameters were summarized into one number by the use of the “anticoccidial index” (Anonymous 1960; McManus et ~2. 1968). A brief description of the derivation of the index follows: The anticoccidial index is the sum of (1) the percentage survival plus (2) the percentage relative weight gain minus (3) the lesion index, and minus (4) the oocyst index. Each of the contributing components of the anticoccidial index was computed as follows: (1) The percentage survival (O100) contributes the corresponding number of points (o-100). (2) The percentage relative weight gain, calculated by dividing the weight gain of the group by the average weight gain of the noninfected, nonmedicated Ygrouns, and multinlied bv, A. L
ET
AL.
100 (to convert to percentage) contributes 0 to 100 points or more or less. (3) The observed average lesion score (04.0) is multiplied by 10 to become the corresponding lesion index (040). (4) The oocyst count of a test group is divided by the mean oocyst count of the infected, nonmedicated groups, then multiplied by 100 (to obtain an observed percentage of contro1). This number is muhiphed by 0.4 to become the assigned oocyst index. The oocyst index is 0 to a maximum of 40 except in the case of the infected, nonmedicated control groups. In this latter instance the index for in&vi&al infected, nonmedicated groups may be more or less than 40 but the mean index for these groups is always 40. Therefore survival and weight gain are each assigned equal importance and gross lesions and oocyst counts are assigned equal importance. Weight gains and survival each contribute 2.5 times the numerical value of the gross lesions and oocyst counts to the anticoccidial index. An anticoccidial index reresponse of 180 or higher for a medication is rated good. Two experimental designs were used to test for reversibility of anticoccidial activity of t-butylaminoethanol by choline. In the first and simpler design, 0.05% t-butylaminethanol (the minimal effective level) was tested alone and combined with a range of levels of choline. In the second experimental design, the level of t-butylaminoethanol was decreased by the use of compounds that are synergistic with it. As stated above, t-butylaminoethanol is synergistic with sulfaquinoxaline against E. tenellu. Sulfaquinoxaline is synergistic with pyrimethamine (Lux 1954; KendaII and Joyner 1956). The same two designs were also used for efficacy reversal experiments with dimethylaminoethanol. Tolerance For evaluation of toxicity of the test compounds in young chickens, battery tol-
Eimeria
tenella:
TABLE Efficacy of t-Butylaminoethanol Dietary LButylaminoethanol HCl
Methylaminoethanol HCl
(%)
(%)
N0lle
NOIE (Noninfected,
NOIX
NOW (Infected.
against Eimeriu
I
tenek Laboratory Strain-18, and Efficacy Reversal Parameter
No. birds
medication
Mortality
Choline chloride
Dimethylaminoethanol HCl
15
CHOLINE REVERSAL
(%)
(%)
Relative weight gain
Average lesion score
(%)
(%) NOIE nonmedicated NOlIt nonmedicated NOLIe NOIE NOIE
Basal* controls) Basal controls) Basal Basal Basal
Average No. oocysts/ bird x 10’
Anticoccidid index
40
0
100
0.0
0.0
200
40
54
54
3.8
5.6
22
20 20 20
0 0 0
60 80 86
3.8 2.2 0.2
6.4 2.8 0.0
82 138 184
20 20 10
0 0 0
94 98 91
0.2 1.1 0.8
0.5 3.4 1.8
188 163 170
0.0125 0.025 0.05
NOW NOlIe NOnI?
0.05 0.05 0.05
NOIll NOIE NOIE
NOW. NOM NOW3
Basal Basal Basal
0.05 0.05 0.05
NOW NIXE NOIE!
0.025 0.05 0.1
BSSSl Basal Basal
20 20 10
0 0 0
88 91 90
0.5 1.6 3.2
0.7 1.8 10.6
178 162 118
0.05
0.05
NOlIt?
BE&Sal
10
0
89
0.0
0.1
188
a Basal
diet
contained
0.267%
choline
+ 0.5 + 1.0 + 2.0
Cl.
erance tests in noninfected birds were performed which continued from 4 to 22 days of age. In these experiments, dayold white cross birds were placed in electrically heated wire-floored batteries in a temperature-controlled room and fed the basal nonmedicated ration for 3 days. At 4 days of age the chickens were weighed and sorted into groups with 2-g weight increments, They were randomly assigned to cages containing four to eight birds with similar weight distributions and total weights. Group weights were taken at 6 and 12 days on test, and individual weights were taken at the termination of the experiment on test Day 18. The same basal diet was used throughout, with the exception of one tolerance experiment. The diet for the one tolerance experiment contained 0.188% choline chloride instead of 0.267% choline chloride. Choline analyses were conducted by the Wisconsin Alumni Research Foundation, Madison, Wisconsin. Choline bitartrate was used for dietary supplementation to the choline chloride already present in the basal diet because it is one of the least hygroscopic salts of choline. Weights of supple-
mental choline bitartrate were corrected to give molar choline chloride equivalents and all choline dietary concentrations appearing in the text, tables, and figures indicate the chloride salt. All medicated diets in Tables II and III contained 0.0005% ethopabate. Hydrochloride salts of t-butylaminoethanol and dimethylaminoethanol were used in all experiments and dietary concentrations appearing herein are of the HCI salts. RESULTS
Anticoccidial
Eficacy
A dietary level of 0.05% t-butylaminoethanol showed good activity against a virulent infection of Eimeriu tenella (Table I). Choline, 1.0 and 2.0%, was added to the basal diet, which already contained 0.267% choline, for the choline reversal experiments summarized in Table I. A total of 1.267 to 2.267% choline incompletely reversed the efficacy of t-butylaminoethanol. The choline which is supplemented and the 0.267% which is already in the basal diet will be added together in the presen-
16
MCMANUS TABLE Efficacy
of Synergistic Dietary
ET AL. II
against Eimeria tenella DP-447
Combinations
and Reversal
by Cldinc -
raramcter
medication birds
Sulfaquinoxalino
t-Butylaminoethanol HCl
(%)
Pyrimethamine
Choline chloride
iUortality
(%I
(%)
Relative weight gain
Average lesion score
(‘%I)
(%I
(%) NlXle (Noninfected, NOIE NOWZ (Infected,
NCJIle
NOlli2 nonn~edicated None nonmedicated
Average NO. ooeysts/ bird X IO”
Anticoccidisl index
Basal* controls) Basal controls)
20
0
100
0.0
0.0
200
50
30
G3
4.0
37.3
53
N0ne None
O.OOG 0.006
0.0003 0.0005
Basal Basal
10 10
30 10
83 8X
2.7 2.6
34.9 29.2
91 121
0.01 0.015 0.02
0.006 0.006 0.006
0.0003 0.0003 0.0003
&Ma1 Basal Basal
10 10 10
0 0 0
95 87 94
2.5 1.4 0.4
12.0 6.9 1.1
157 lG7 189
0.01 0.015 0.02
0.006 0.006 0.006
0.0005 0.0005 0.0005
10 10 10
0 0 0
94 9G 87
0.X 0.3 0.4
I.5 1.9 2.5
184 191 180
0.01 0.015 0.02
0.006 O.OOB 0.006
0.0003 0.0003 0.000~
Basal Basal Basal
+ 0.5 + 0.5 + 0.5
10 10 10
0 0 0
09 91 97
1.8 1.9 0.8
8.1 0.5 7.0
172 16s 181
0.01 0.015 0.02
0.006 0.006 0.006
0.0005 0.0005 0.0005
Rassl Basal Basal
+ 0.5 + 0.5 + 0.5
10 10 10
0 0 0
98 98 89
0.9 0.0 0.2
1.4 0.0 0.0
IS7 198 187
0.01 0.015 0.02
0.006 0.006 O.OOG
0.0003 0.0003 0.0003
Ram1 Basal nasal
+ + +
1.0 1.0 1.0
10 10 10
0 0 0
94 92 94
2.1 1.1 1.2
1G.S 4.0 4.9
1x 177 177
0.01 0.015 0.02
0.006 0.006 0.006
0.0005 0.0005 0.0005
Bass1 Basal Basal
+ + +
1.0 1.0 1.0
10 10 10
0 0 0
92 93 97
1.5 I.4 0.5
15.2 7.8 1.3
161 171 191
0.01 0.015 0.02
0.006 0.006 0.006
0.0003 0.0003 0.0003
Basal Basal Basal
+ 2.0 + 2.0 + 2.0
10 10 10
20 0 0
80 GO 8ti
3.5 3.1 1.2
2’S.0 32.2 8.7
95 103 lfi.5
0.01 0.015 0.02
0.006 0.006 0.006
0.0003 0.0005 0.0005
Basal Basal Basal
+ 2.0 + 2.0 + 2.0
10 10 10
10 0 0
80 7s 88
2.!) 1.9 1.1
11.8 9.2 12.5
134 149 164
0 Basal
diet
contained
0.267%
choline
Cl.
tation. The levels appearing in the text, tables, and figures are the total dietary concentration. In the second efficacy protocol (Materials and Methods) synergistic combinations were used. t-Butylaminoethanol is synergistic with sulfaquinoxaline against E. tenella. Sulfaquinoxaline is synergistic with pyrimethamine. A mixture of 0.006% sulfaquinoxaline, 0.0003 to 0.00057, pyrimethamine, and 0.01 to 0.02% t-butylaminoethanol was highly effective in chickens given heavy exposure to E. tenella (Tables II and III).
Choline reversal experiments were conducted with sulfaquinoxaline and pyrimethamine combined with a range of levels of t-butylaminoethanol from 0 to 0.02% (Table II). Choline, 2.267%, totally reversed the anticoccidial effect contributed by 0.01% t-butylaminoethanol in synergistic combination with 0.0067, sulfaquinoxaline and 0.0003 to 0.00057, pyrimethamine. Based on all parameters of the experiment, the reversing effect of 2.267% choline was such that there was little difference in response to 0.006% sulfaquinoxaline plus pyrimethamine with or without 0.01% t-butylaminoethanol.
Eimeria tenda:
CHOLINE
TABLE
III
Efficacy of Synergistic Combinations against Eimeria and Reversal by Dimethylaminoethanol Dietary t-Butylaminoethanol HCI
Sulfaquinoxaline (%)
medication Pyrimethamine (%)
tenella DP-447 Parameter
No. hirds Dimethylsminoethanol HCl
17
REVERSAL
MOP tality (%)
Relative weight gain
Average lesion score
(%)
(%)
(%) NOIE
NOI%2
(Noninfected, NOW
(Infected,
NOIll?
nonmedicated NOIll? NOW nonmedicated
Average NO. oocysts/ bird x 10”
Anticoccidial index
NOW
20
0
100
0.0
NOW
50
38
68
4.0
27.6
50
NOW
0 0 0
103 99 97
1.1 0.5 0.0
3.0 3.7 0.0
188 189 197
controls) controls)
0.01 0.015 0.02
0.006 0.006 0.006
0.0003 0.0003 0.0003
NOW
10 10 10
0.01 0.015 0.02
0.006 0.006 0.006
0.0005 0.0005 0.0005
None NOW None
10 10 10
0 0 0
98 97 106
0.8 0.0 0.0
3.7 0.0 0.0
185 197 206
0.01 0.015 0.02
0.006 0.006 0.006
0.0003 0.0003 0.0003
0.05 0.05 0.05
10 10 10
0 0 0
9.5 107 101
1.2 0.8 0.4
11.8 3.8 0.8
166 193 196
0.01 0.015 0.02
0.006 0.006 0.006
0.0005 0.0005 0.0005
0.05 0.05 0.05
10 10 10
0 0 0
104 99 103
0.7 0.2 0.6
2.9 0.8 4.0
193 196 191
0.01 0.015 0.02
0.006 0.006 0.006
0.0003 0.0003 0.0003
0.1 0.1 0.1
10 10 10
10 0 0
95 96 82
2.5 3.1 1.5
12.0 36.1 6.2
143 125 158
0.01 0.015 0.02
0.006 0.006 0.006
0.0005 0.0005 0.0005
0.1 0.1 0.1
10 10 10
0 0 0
104 90 101
1.2 1.8 0.2
7.6 3.3 1.0
181 167 198
0.01 0.015 0.02
0.006 0.006 0.006
0.0003 0.0003 0.0003
0.2 0.2 0.2
10 10 10
0 10 10
68 85 86
3.2 3.1 3.3
13.7 22.2 25.2
116 112 106
0.01 0.015 0.02
0.006 0.006 0.006
0.0005 0.0005 0.0005
0.2 0.2 0.2
10 10 10
10 0 0
96 99 87
2.4 2.2 2.0
23.1 12.8 11.2
129 158 151
NOIE
Dimethylaminoethanol, O.l%, brought about a substantial but incomplete reversal of the anticoccidial action of 0.05% t-butylaminoethanol (Table I), Methylaminoethanol had no effect in reversal experiments with t-butylaminoethanol (Table I). Dimethylaminoethanol, 0.27~~ totally reversed the anticoccidial activity contributed by 0.01% t-butylaminoethanol in the synergistic combination with 0.006% sulfaquinoxaline and 0.0003 to 0.0005% Dimethylaminoethanol, pyrimethamine. 0.2%, sometimes but not consistently reversed 0.015 to 0.02% t-butylaminoethanol in the synergistic combination. Levels
below 0.2% had less reversing effect (Table III). Noninfected, medicated control groups of birds were tested with the highest dietary levels of the mixtures of chemicals used in the reversal experiments of Tables II and III. These tests were toxicity tests which followed the protocol of the efficacy assays. They are not to be confused with the more precise tolerance tests in chicks which ran from 4 to 22 days of age. The tests with noninfected, medicated controls were necessary to assure that toxicity of the several chemicals was not affecting the efficacy results. The mixtures
1s
MCMANUS
Sulfaquinoxaline (70)
Choline chloride (5%)
t-Butyl aminoethanol HCl (%I
Pyrimethamine cm
None None None None None
None None None None None
None None None None None
Basala Basal Basal Basal Basal
0.01
0.0003
0.006
0.01
0.0003
0.006
0.015 0.015 0.02 0.02
0.0003 0.0003 0.0003 0.0003
0.006 0.006 0.006 0.006
Basal Basal Basal Basal Basal Basal
0.02 0.02 0.02 0.02
0.0003 0.0003 0.0003 0.0003
0.006 0.006 0.006 0.006
Basal Basal Basal Basal
= Basal diet contained
ET
AL.
Dimethylaminoethanol HCl (75)
Wright gain Cd
270 278 271 267 285 + + + + + +
2.0 2.0 2.0 2.0 2.0 2.0
279 256 2.58 267 259 270 282 280 279 271
0.2677{; choline Cl.
tested did not significantly depress weight gain (P Q 0.05, Table IV). Tolerance Eighteen-day tolerance experiments demonstrated marked toxicity with high mortality for 0.04 and O.OScjo t-butylaminoethanol. Toxicity was not well defined in the shorter (g-day) efficacy tests which ran from 14 to 23 days of age. Eighteen-day tolerance tests were performed in one experiment with a commercial diet containing 0.188% choline. Weight gain is plotted against graded concentrations of t-butylaminoethanol (Fig. 1). The dose-response curve intercepted zero growth depression at 0.019% t-butylaminoethanol. Details of the experiment are presented in the legend of Fig. 1. A relationship of choline to tolerance by the chicken of t-butylaminoethanol re-
mained to be established. In further tolerance experiments, diets were supplemented with a fixed toxic level of t-butylaminoethanol, and choline was made the variable. Weight gain is plotted against (Figs. 2 graded choline concentrations and 3). In tests with 0.04% t-butylaminoethanol, the intercept for zero growth depression is 0.451% choline (Fig. 2). In tests with diets supplemented with 0.05% t-butylamilloethanol, the intercept is 0.513c/o choline (Fig, 3). Mortality due to t-butylaminoethanol was prevented by choline (Figs. 2 and 3). Details of the experiments are presented in the legends of the figures. Similary designed toxicity-reversal exdimethylaminoethanol periments with clearly demonstrated that it, as well as choline, was capable of reversing the adverse effects of t-butyaminoethanol on growth and survival of young chickens. The toxicity of 0.04 and 0.05% t-butyl-
Eimeria
tenella:
19
CHOLINE REVERSAL
I IO-
100 -
. .
50 IMortality:
4%
0%
4%
I 0.01
I
I
I
0.02
0.03
0.04
Percent
Dietary
t-Butylaminoethanol
2 I%
HCl
FIG. 1. Effect of feeding graded levels of t-butylaminoethanol upon the growth of chickens from 0.5 to 3 weeks of age. Each dot represents the relative percentage weight gain of one pen of four chickens. The horizontal lines represent the average weight gain (solid line) and the 0.95 probability confidence interval (dashed lines) of 20 pens of nonmedicated control chickens ( 100% = 362.1 g f 3.7%). The sloping lines are the dose-response curve (solid line) and its 0.95 probability confidence interval (dashed lines) for the growth response of 18 pens of chickens fed 0.02 to 0.04% t-butylaminoethanol HCl. The equation for the doseresponse curve is y = -52.99 - 88.96x where x = log percentage dietary t-butylaminoethanol HCl and y = relative percentage weight gain. In the calculation of linear regression, the responses were weighted to reflect mortality in some of the pens.
aminoethanol was incompletely reversed by 0.1% dimethylaminoethanol, the highes2 level tested. Mortality was totally prevented and weight gain was markedly improved but not fully returned to norma1 (Table V). DISCUSSION
The anticoccidial efficacy and the adverse effects on growth and survival of young chickens of t-butylaminoethanol are reversible by choline and by dimethylaminoethanol. The ratios of the dose of inhibitor to metabolite for reversal can be approximated. Unusually large quantities of choline were required for anticoccidial reversal of
t-butylaminethanol. A dietary level of 0.05% t-butylaminoethanol was incompletely reversed by 2.267% choline (Table I) giving a reversal ratio of 1 part inhibitor to > 45 parts metabolite. Although the 2.267% level of choline was not toxic, it was probably close to a growth depressing dietary concentration (Melass et al. 1946), and efficacy reversal experiments with dietary levels of choline higher than 2.267% were not attempted. Synergistic mixtures of t-butylaminoethano1 with suIfquinoxaline and pyrimethamine permitted lower medication levels of t-butylaminoethanol (Table II). In the synergistic combination, the anticoccidial contribution of 0.010/o t-butylaminoethanol was reversed by
20
MCMANUS
ET
AL.
t 100
t
60
0.267
0.367 Percent
Dietory
0.467
Of i67
Choline Cl
FIG. 2. Effect of graded levels of dietary choline Cl fed in combination with 0.04% t-butylaminoethanol HCl upon the growth of chickens from 0.5 to 3 weeks of age. Each dot represents the relative percentage weight gain of one pen of eight chickens. The horizontal line represents the average weight gain (solid line) and its 0.95 probability confidence interval (dashed lines) of 12 pens of nonmedicated control chickens ( 100% = 292.6 g 2 4.9%). The sloping lines are the dose-response curve (solid line) and its 0.95 probability confidence interval (dashed lines) for the growth response of 17 pens of chickens fed 0.267 to 0.467% choline Cl in combination with 0.04% t-butylaminoethanol HCl. The equation for the curve is y = 132.69 + 94.42x where x = log percentage dietary choline Cl and y = relative percentage weight gain. In the calculation of linear regression, the responses were weighted to reflect mortality in scme of the pens.
2.267% choline, giving a reversal ratio of about 1 part inhibitor to 200 parts metabolite. The anticoccidial activity of O.OSY, t-butylaminoethanol was substantially but incompletely reversed by 0.1% dimethylaminoethanol (Table I) indicating a reversal ratio of 1 part inhibitor to > 2 parts metabolite. In further antiparasitic reversal experiments with dimethylaminoethanol employing the synergistic mixture of t-butylaminoethanol, sulfaquinoxaline, and pyrimethamine (Table III), the anticoccidial contribution of 0.01% t-butylaminoethanol was reversed by 0.2% dimethylaminoethanol which indicates a
reversal ratio of approximately 1 part inhibitor to 20 parts metabolite. For calculation of the above ratio, the contribution of the choline in the basal diet to reversal of the anticoccidial action by dimethylaminoethanol is relatively unimportant because the anticoccidial reversal ratio of t-butylaminoethanol to choline is unusually low. The toxicity for the chicken of 1 part t-butylaminoethanol was overcome by approximately 10 parts dietary choline. Levels of 0.0191, 0.01, and O.OSY, t-butylaminoethanol were tolerated by the young chicken when choline levels were, respectively, 0.188, 0.451, and 0.513r/o. The lin-
Eimeriu
tenella:
110
CHOLINE I
I -s-m
21
REVERSAL I
l
2%
0%
l l
l
100
l
60 Mortolity:
15%
50% I
I
I
I
0.267
0.367
0.467
0.66 7
Percent
Dietory
Choline Cl
FIG. 3. Effect of graded levels of dietary choline Cl fed in combination with 0.05% t-butylaminethanol HCl upon the growth of chickens from 0.5 to 3 weeks of age. Each dot represents the relative percentage weight gain of one pen of eight chickens. The horizontal line represents the average weight gain (solid line) and its 0.95 probability confidence interval (dashed lines) of 12 pens of nonmedicated control chickens ( 100% = 292.6 g f 4.9%). The sloping lines are the dose-response curve (solid line) and its 0.95 probability confidence interval (dashed lines) for the growth response of 18 pens of chickens fed 0.267 to 0.467% choline Cl in combination with 6.05% t-butylaminoethanol HCl. The equation for the doseresponse curve is y = 118.21+ 62.81~ where x = log percentage dietary choline Cl and y = relative percentage weight gain. In the calculation of linear regression, the responses were weighted to reflect mortality in some of the pens.
ear relationship of the experimentally determined ratios of inhibitor to metabolite is presented graphically in Fig. 4. The correlation coefficient (T) for linearity is 0.99. Additional evidence for linearity is the fact that the extrapolated curve is close to intercepting x = 0, y = 0. This is expected. Toxicity for the chicken of 0.04 and 0.05% t-butylaminoethanol was almost totally reversed by 0.1% dimethylaminoethanol. Weight gain was markedly improved but not returned completely to normal. Mortality was totally prevented (Table V). For calculation of the toxicityreversal ratio of t-butylaminoethanol to dimethylaminoethanol, the contribution to
reversal by the choline in the basal diet (0.267%) is important. Figure 4 indicates that 0.267% choline reverses toxicity of approximately 0.0257, t-butylaminoethanol. In the basal diet supplemented with 0.04% t-butylaminoethanol, a 0.025% portion of the 0.04% inhibitor is reversed by the choline in the basal diet giving a difference of 0.0470 - 0.025% = 0.015%. For reversal of toxicity for the chicken, the inhibitor to dimethylaminoethanol ratio is approximately 0.015: 0.1 or approximately 1:7. A singular feature of the results presented is the large amount of dietary metabolite required for reversal of t-butylaminoethanol. The quantity of choline
22
MCMANUS
I
0.05 E
B
2 5 f Q % m A 6 .-t 0 E g a”
ET
I
AL.
I
I
I
0.04
0.03
0.02 t
0.01
I
0
0
0. 6 Percent
Dietary
Choline
Cl
FIG. 4. Tolerance of t-butylaminoethanol with increasing levels of choline. Each dot represents the concentration of choline required to overcome growth depressing effects in the chicken of a level of t-butylaminoethanol. The equation for the curve is y = 0.00163 -I0.09046x where x = percentage dietary choline Cl and y = percentage dietary t-butylaminoethanol HCI.
TABLE Reversal
of Toxicity
Weight
of t-Butylaminoethanol
gain, relative
percentage Dietary
Expt No.
None
A. Diets supplemented
V by Dirnethylaminoethanol
of nonmedicated
percentage
control
dimethylaminoethanol
0.025
with 0.04y0 t-butylaminoethanol
chickens HCI
0.05
0.1
HCl
1
87.0 (7/8)G 73.3 (6/S)
87.6 (S/S) 83.4 (7/8)
92.4 (8/S) 101.9 (8/8)
97.7 (8/S) 93.5 (8/8)
2
64.7 (6/S) 51.7 (2/8)
67.4 (7/8) 87.4 (7/S)
73.3 (7/8) 8.5.2 (S/S)
78.4 (8/8) 88.6 (8/8)
69.2
81.4
88.2
89.6
9
3
0
Average Mortality
(72)
34
B. Diets supplemented
with 0.05’s t-butylaminoethanol
HCl
1
77.0 (5/S) 70.3 (S/8)
85.8 (5/8) 100.5 (3/S)
101.5 (8/8) 87.8 (8/8)
90.4 (S/8) 86.6 (8/8)
2
54.4 (S/8) 40.7 (l/S)
70.3 (7/S) 87.1 (3/S)
77.3 (S/B) 92.3 (6/8)
80.7 (S/8) 87.0 (8/8)
60.6 62
85.9 44
89.7 12
86.2 0
Average Mortality (1Number
(70)
in parentheses
is number
of survivors
in each pen of eight birds.
Eimeria
tenekz: CHOLINE
required for reversal of the antiparasitic effect is especially large. Choline is a quaternary cation and therefore is totally ionized in solution. Dimethylaminoethanol is a tertiary amine (pK, 9.26). At pH 7.4, approximately is non1.4% of dimethylaminoethanol ionized. This difference between quaternary nitrogen compounds and tertiary amines has important implications for diffusion across biological membranes. These membranes generally are lipid in nature and nonionized compounds diffuse across such barriers at a more rapid rate than ionized compounds. Movement of choline across membranes of the parasite may be restricted because of its ionization properties. This could relate to the large amount of choline required by the parasite for reversal of the inhibitor. A much smaller amount of choline is required for reversal of toxicity of t-butylaminoethanol for the chicken than for the parasite. In other words there is a marked species specificity for the reversal by choline. Obviously, a favorable therapeutic ratio is achieved with a compound whose toxicity is more easily overcome for the host than for the parasite.
23
REVERSAL
REFEREN1960. Technical Bulletin, AMPROL, an effective new coccidiostat for broilers. Merck and Co., Inc., Rahway, New Jersey. JOHNSON, J., AND REID, W. M. 1970. Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments with chickens. Experimental Parasitology 28, 30-36. KENDALL, S. B., AND JOYNER, L. P. 1956. The potentiation of coccidiostatic drugs by pyrimethamine. VeterinuTy Record 68, 119-121. Lux, R. E. 1954. The chemotherapy of Eimeria tenella: I, Diaminopyrimidines and dihydroAntibiotics and Chemotherapy 4, triazines. 971-977. MCMANUS, E. C., CAMPBELL, W. C., AND CUCKLER, A. C. 1968. Development of resistance to quinoline coccidiostats under field and laboraJournal of Parasitology 54, tory conditions. 1190-1193. MCMANUS, E. C., AND ROGERS, E. F. 1969. Anticoccidial effect of t-butylaminoethanol, its reversibility by dimethylaminoethanol and its synergistic interaction with sulfonamides. Federation Proceedings 28, 390. MCMANUS, E. C., TAMAS, T., JUDITH, F. R., AND ROGERS, E. F. 1975. Anticoccidial and growth depressing effects in chicks of t-butylaminoethanol and reversibility by choline and precursors. Poultry Science 54, 1793. MELASS, V. H., PEARSON, P. B., AND SHERWOOD, R. M. 1946. Toxicity of choline in the diet of growing chickens. Proceedings of the Society of Experimental Biology and Medicine 62, 174176. ANONYMOUS.