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Enhancement of the Anticoccidial Activity of Polyether Antibiotics in Chickens by Tiamulin
Enhancement of the Anticoccidial Activity of Polyether Antibiotics in Chickens by Tiamulin J. G. MEINGASSNER, F. P. SCHMOOK, R. CZOK, and H. MIETH Sandoz Forschungsinstitut, Brunnerstrasse 59, A-1235 Vienna, Austria (Received for publication August 7, 1978)
1979 Poultry Sci 58:308-313 INTRODUCTION T h e anticoccidial activity of monensin stimulated such interest in polyether antibiotics t h a t m o r e t h a n 2 0 distinct c o m p o u n d s of this class have been reported during t h e last few years (Westley, 1 9 7 5 ; Weppelman etal., 1 9 7 7 ) . Several are n o w widely used alone or in combination with o t h e r drugs as p r o p h y l a c t i c agents against coccidiosis ( D a m r o n etal., 1 9 7 7 ; Mitrovic etal., 1 9 7 7 ; Mitrovic and Schildknecht, 1975a,b) or as feed additives in r u m i n a n t s (Perry et al, 1 9 7 6 ; Raun et al., 1 9 7 6 ) . Tiamulin (Tiamutin®) is a new pleuromutilin derivative (Drews et al., 1 9 7 5 ) soon t o be launched as a prophylactic and t h e r a p e u t i c drug against m y c o p l a s m a infections in p o u l t r y (Laber a n d Schiitze, 1 9 7 5 ) and pigs (Stipkovits etal., 1 9 7 8 ) . In the c o n t e x t of an evaluation of its compatibility with o t h e r drugs l a b o r a t o r y investigations were u n d e r t a k e n t o s t u d y t h e anticoccidial efficacy of m o n e n s i n and lasalocid in t h e presence of tiamulin. The present comm u n i c a t i o n reports t h e results of these experim e n t s and indicates t h a t although tiamulin alone is inactive, its c o m b i n a t i o n with b o t h polyether antibiotics enhances their anticoccidial activity. These findings were postulated t o result from a metabolic interaction b e t w e e n these c o m p o u n d s in t h e host. In order t o evaluate t h e t y p e of interaction, e x p e r i m e n t s were carried o u t using isolated, perfused rat
liver t o s t u d y t h e metabolic fate of monensin in t h e presence of tiamulin. MATERIALS AND METHODS Experiments in Chickens. Material and experimental procedures used in t h e present s t u d y have already been described (Meingassner et al, 1977). The e x p e r i m e n t s were c o n d u c t e d in batteries with day old broiler t y p e chickens inoculated with E. tenella at 4 days of age (day 0). The medicated feed was fed t w o days before infection (day —2) and c o n t i n u e d until t h e end of t h e e x p e r i m e n t s on day +7 (Experim e n t 1, 2, 4 ) or +8 ( E x p e r i m e n t 3). Chickens in E x p e r i m e n t 5, which was t e r m i n a t e d on day +14, were given n o n m e d i c a t e d feed from day +7 onwards. Tiamulin was administered via t h e drinking water for three days from t h e t i m e of infection. Measured parameters included mortality due t o coccidiosis, fecal score, lesion score, weight gain, and percentage weight gain (Experiments 1, 2, 3, 4) or oocyst o u t p u t ( E x p e r i m e n t 5) of t r e a t e d chicks in comparison t o infected, u n t r e a t e d or uninfected, u n t r e a t e d controls. F u r t h e r details of individual experiments and medication levels are given in t h e tables or figures. Biochemical studies. Rat Liver Perfusion. Livers of female Sprague Dawley rats (200 t o 2 5 0 g) were perfused using t h e m e t h o d s of Mitzkat ( 1 9 6 8 ) and Schimassek ( 1 9 6 3 ) with
308
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ABSTRACT The anticoccidial activities of monensin and lasalocid have been studied separately and in combination with tiamulin, a new pleuromutilin derivative. Combinations of constant tiamulin concentration (.0125%) in drinking water with various levels of polyether anticoccidials (6.3 to 125 ppm) in feed and conversely of constant levels of anticoccidials with various concentrations of tiamulin were used. The prophylactic efficacy of these combined treatments in battery raised broiler chickens infected with Eimeria tenella was evaluated. Assessment of the parameters mortality, weight gain, dropping scores, lesion scores, and oocyst output showed that simultaneous application of tiamulin significandy improved the anticoccidial activity of the polyethers. As tiamulin alone is without anticoccidial activity, this phenomenon was considered to result from an interaction between tiamulin and the polyethers leading to a slower metabolic degradation of the latter. Thus tissue levels adequate for maximum anticoccidial activity would be attained with lower polyether dose levels. Experiments using isolated perfused rat liver showed that elimination of monensin was reduced by 60% in the presence of tiamulin.
TIAMULIN AND POLYETHER ANTIBIOTICS
RESULTS AND DISCUSSION
Parasitological Data. Table 1 summarizes Experiment 1 in which the effect of monensin or tiamulin alone was compared with their efficacy in combination. Monensin at 125 ppm prevented mortality and largely suppressed clinical signs and the appearance of lesions in comparison to untreated controls. Tiamulin at .0125% in the drinking water (a dose calculated to provide 25 to 30 mg/kg body weight) exhibited no anticoccidial activity, and treated animals were as severely affected as the infected controls. When monensin at levels of 25 ppm to 125 ppm was combined with .0125%
tiamulin, a reduction in fecal score and lesion score was observed compared to 125 ppm monensin alone. Improved responses were observed using 12.5 ppm monensin plus .0125% tiamulin. Feeding monensin levels higher than 50 ppm in combination with .0125% tiamulin depressed body weight. However, lower monensin levels caused a marked improvement in weight gain over that achieved with 125 ppm monensin alone. The effect of monensin and tiamulin alone or in combination on oocyst production is shown in Figure 1. Tiamulin (.0125%) or monensin (12.5 ppm) alone exhibited no marked suppressive effect. However, a combination of both drugs at these concentrations resulted in a reduction of oocyst output to a level lower than that achieved with 125 ppm monensin alone. The anticoccidial activity of lasalocid in combination with a constant tiamulin concentration was studied in Experiment 2. The data are presented in Table 2 and show that lasalocid at 12.5, 25, 50, or 75 ppm plus tiamulin at .0125% resulted in an increase in body weight over the treatment with 75 ppm lasalocid alone. Groups medicated with 25 ppm lasalocid plus .0125% tiamulin exhibited improved fecal score, survival rate, lesion score, and weight gain when compared with lasalocid alone. Weight gain was not decreased by the addition of tiamulin to levels of 75 ppm lasalocid. The anticoccidial activity of the polyethers in combination with reduced tiamulin levels was studied in Experiments 3 and 4. A combination of 125 ppm monensin plus .00063 to .0100% tiamulin resulted in better coccidial control, as indicated by reduced fecal score and lesion score, than either drug alone (Table 3). However, a combination of high concentrations of both drugs depressed body weight as observed in Experiment 1. Better results were achieved when tiamulin was used at concentrations below .0025% in the combined treatments. Recommended levels of lasalocid plus various tiamulin concentrations gave excellent coccidiosis control in terms of weight gain, survival rate, and fecal and lesion score in comparison to lasalocid alone (Table 4). Indeed even 75 ppm lasalocid alone did not completely prevent coccidiosis, although it did control weight gain. However, some groups receiving combined therapy showed as much as 15% weight gain above that achieved with 75 ppm lasalocid alone.
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180 ml of medium (Tyrode, 2.6% bovine albumine and bovine erythrocytes with total of 10 g% hemoglobin). Thirty minutes after starting the perfusion, tritiated tiamulin (3.7 juCi/mg) and/or monensin (.4, 1, or 2 mg dissolved in pure dimethylsulfoxide (dest. in vacuo)) were added. Samples of the medium (2 to 4 ml) were taken at specified times during the 3 hr perfusion. The excreted bile was collected for determination of radioactivity and for thin layer chromatography. Thin Layer Chromatography (TLC). Medium samples were centrifuged and the proteins of the supernatant collected by recentrifugation after precipitation with 20 times the volume of A-grade methanol. This extraction of the precipitate was repeated twice. The methanol-phase was evaporated, the residue dissolved in methanol and chromatographed on TLC plates (Merck No. 5737). The plates were developed and sprayed according to Golab et al. (1973). Bile was chromatographed without any previous preparation. The densitometric determination of monensin was carried out with a Schoeffel spectrodensitometer at 515 nm. The amounts of monensin in the samples were determined by means of appropriate standards on each plate. The radioactivity of the samples was determined in a Packard Tri-Carb liquid scintillation counter M 3395. Drugs. Monensin, provided by Messrs. Kwizda, Vienna, was used in pure form for in vitro studies and/or as premix (Elancoban) in chickens. Lasalocid was obtained in pure form (lot No. 1804-114) from Hoffman-La Roche, Basle. A soluble powder of tiamulin (45%, lot No. KRV 310-H/HA2) was used for administration via the drinking water.
309
310
MEINGASSNER ET AL.
TABLE 1. Anticoccidial efficacy of monensin and tiamulin separately or in combination at constant tiamulin concentration against E. tenella in the chick up to 7 days after infection3-*0^ (Experiment 1) Ppm in feed
Drugs Monensin Tiamulin Monensin + Tiamulin Monensin + Tiamulin Monensin + Tiamulin Monensin + Tiamulin Monensin + Tiamulin Monensin + Tiamulin Infected controls Non-infected controls
%in drinking water
125
+ + + + + 12.5 +
125 100 75 50 25
.0125 .0125 .0125 .0125 .0125 .0125 .0125
FS
S%
LS
WG
WG%
3.5 6.0 0 0 0 0 1 1 6.5 0
100 92 100 100 100 100 100 100 67 100
.9 2.7 0 0 .25 0 1.0 1.7 2.6 0
78.6 79.7 54.9 62.1 73.2 81.0 83.7 86.9 75.2 91.8
85.6 86.8 59.8 67.6 79.7 88.2 91.1 94.7 81.9 100.0
FS: Fecal score, S%: Survival rate; LS: Lesion score; WG: Weight gain; WG%: Percentage weight gain. b 2 X 6 chicks per group, inoculum: 5.104 oocysts, strain Hoe. Treatment: Monensin in feed from day —2 to +7, tiamulin in drinking water from day 0 to +3.
Tiamulin alone has no anticoccidial activity. Thus its in vivo augmentation of the efficacy of the polyethers is not comparable with the synergistic effects demonstrated between 4hydroxyquinoline and pyridine coccidiostats (Challey and Jeffers, 1973) or clopidol and methylbenzoquate (Ryley, 1975). Neither does tiamulin alone, or in combination with polyethers, act indirectly by an inhibition of the
• A V
107H
^i^ •* 1
* •
monensin 125 ppm monensin 12.5 ppm tiamulin 0.0125 1. tiamulin 0.0125 "'• monensin 12.5 ppm infected controls
10 6
10 5 -
10 4 -
//— 6
7 8 9 10 11 Days after inoculation
12
13
14
FIG. 1. The effect of monensin and tiamulin separately or in combination on the oocyst output of chickens infected with E. tenella (Experiment 5). 2 X 6 chicks per group, inoculum: 5.103 oocysts, strain Hoe. Treatment: monensin in feed (—2 -> 7 days), tiamulin in drinking water (0 -* 3 days).
excystation process (measured by intact oocysts recovery from the feces in the 24 hr following inoculation) or by alteration of gut mobility (Meingassner, unpublished data). Thus it is probable that the enhancement of the anticoccidial activity of the polyether antibiotics by tiamulin results from some interactions with the hosts' metabolism. Biochemical data. As a direct test of this proposition, the metabolic fate of monensin and tiamulin separately or in combination were monitored using isolated, perfused rat liver (IPL). Addition of 2 mg of monensin to IPL markedly altered its functions. Temporary swelling occurred in conjunction with a lower perfusion rate, while an elevation of potassium levels in the perfusion medium indicated cell damage. The rate of urea production decreased by 50% over a 2 hr period pointing to a lowering of gluconeogenesis, and, indeed, glucose levels did not rise any more. In addition, very low levels of lactate and extremely high levels of pyruvate occurred in the perfusion medium indicating a disturbance in liver redox potential. Less pronounced, but still observable signs of liver disturbance (e.g. the rate of urea production was reduced for 1 hr) were produced by 1 mg of monensin. However, .4 mg of monensin only reduced urea production for 30 min. It was thus considered to be a tolerable dose for the liver and proved sufficient for the study of the kinetics of monensin elimination from the perfusion medium. The polyether was elimi-
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a
TIAMULIN AND POLYETHER ANTIBIOTICS
311
TABLE 2. Anticoccidial efficacy of lasalocid and tiamulin separately or in combination at constant tiamulin concentration against E. tenella in the chick up to 7 days after infection2-*®'>c (Experiment 2)
Drugs Lasalocid Tiamulin Lasalocid + Tiamulin Lasalocid + Tiamulin Lasalocid + Tiamulin Lasalocid + Tiamulin Lasalocid + Tiamulin Infected controls Non-infected controls
Ppm in feed
% in drinking water
75
.0125 75 +.0125 50 +.0125 25 +.0125 12.5+ .0125 6.3 + .0125
FS
S%
LS
WG
WG%
3.5 8.5 0 0
96 63 100 100 100 92 54 45 100
1.5 2.8 0 .15 1.1 2.8 3.0 2.3 0
86.1 67.7 95.9 93.2 94.9 88.4 77.5 61.0 86.0
100.1 78.7 111.5 108.4 110.4 102.8 90.1 70.9
1.75 4.8 7.5 8.0 0
100
4 X 6 chicks per group, inoculum: 2.10 s oocysts, strain Hoe. Treatment: Lasalocid in feed from day —2 to +7, tiamulin in drinking water from day 0 to +3.
nated via t h e bile at an exponential rate with a half life of 7 min (Figure 2 ) . However, t h e various m e t a b o l i t e s h a d different colors a n d T L C properties, t h u s making quantitative analysis impossible. Up t o 10 mg of H 3 tiamulin was well tolerated b y t h e liver with n o n e of t h e above signs of i m p a i r m e n t . App r o x i m a t e l y 4 0 % of a 3 mg dose was m e t a b olized within 2.5 hr and excreted via t h e bile. When 3 mg of tiamulin was given s i m u l taneously with .4 mg of monensin t h e elimina-
tion rate of t h e l a t t e r was reduced resulting in a half life of 17 min. T h e excretion of m e t a b olites via t h e bile also proceeded m o r e slowly. Under these conditions t h e signs of incompatibility observed (elevation of potassium, high lactate, and p y r u v a t e levels in t h e perfusion m e d i u m and a lower rate of urea p r o d u c tion for 75 min) were c o m p a r a b l e with those for 1 mg of monensin alone. During this period t h e monensin c o n c e n t r a t i o n in t h e perfusion m e d i u m exceeded t h a t in e x p e r i m e n t s
TABLE 3. Anticoccidial efficacy of monensin and tiamulin separately or in combination at constant monensin concentration against E. tenella in the chick up to 8 days after infection3-*"*'1 (Experiment 3)
Drugs Monensin Tiamulin Monensin + Tiamulin Monensin + Tiamulin Monensin + Tiamulin Monensin + Tiamulin Monensin + Tiamulin Monensin + Tiamulin Monensin + Tiamulin Infected controls Non-infected controls
Ppm in feed
% in drinking water
125
125 125 125 125 125 125 125
.0125 + .0100 +.0075 + .0050 + .0025 + .00125 + .00063 + .00031
FS
S%
LS
WG
WG%
4.3 8.0 0 0 1.7 1.7 3.0 2.7 4.0 6.0 0
100 94 100 100 100 100 100 100 94 94 100
1.3 2.2 0 .1 .5 .1 .7 .3 .7 1.9 0
102.5 79.2 81.1 80.1 86.1 89.4 104.9 109.9 109.0 81.3 109.4
93.7 72.4 74.1 73.2 78.7 81.7 95.9 100.4 99.6 74.3 100.0
FS: Fecal score, S%: Survival rate; LS: Lesion score; WG: Weight gain; WG%: Percentage weight gain. 3 X 6 chicks per group, inoculum: 6.10 4 oocysts, strain Hoe. c
Treatment: Monensin in feed from day - 2 to +8, tiamulin in drinking water from day 0 to +3.
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FS: Fecal score, S%: Survival rate; LS: Lesion score; WG: Weight gain; WG%: Percentage weight gain.
MEINGASSNER ET AL.
312
TABLE 4. Anticoccidial efficacy oflasalocid and tiamulin separately or in combination at constant lasalocid concentration against E. tenella in the chick up to 7 days after infection3-!0'1- (Experiment 4) Ppm in feed
Drugs Lasalocid Tiamulin Lasalocid + Tiamulin Lasalocid + Tiamulin Lasalocid + Tiamulin Lasalocid + Tiamulin Lasalocid + Tiamulin Infected controls Non-infected controls
75 75 75 75 75 75
.0125 + .0075 + .0050 + .0025 + .00125 + .00063
FS
S%
LS
WG
WG%
3.5 8.5 0 0 0 .3 .7 8.0 0
96 63 100 100 100 100 100 45 100
1.5 2.8 0 0 .1 .1 .2 2.3 0
86.1 67.7 96.4 99.9 99.0 99.6 93.8 61.0 86.0
100.1 78.7 112.1 116.2 115.1 115.8 109.1 70.9 100
FS: Fecal score, S%: Survival rate; LS: Lesion score; WG: Weight gain; WG%: Percentage weight gain. 4 X 6 chicks per group, inoculum: 2.10 s oocysts, strain Hoe. Treatment: Lasalocid in feed from day —2 to +7, tiamulin in drinking water from day 0 to +3.
without tiamulin. However, monensin had no effect on the rate of tiamulin elimination. The metabolites found in the plasma were identical with those found without monensin, only the quantities excreted differed slightly. The experiments indicate that simultaneous administration of tiamulin and monensin induces a slower degradation and elimination of the latter from isolated, perfused rat liver. If this response also occurs in chickens, it is likely that the high efficacy of relatively low monensin levels also results from its slower elimination, thus producing tissue levels adequate for maximum anticoccidial activity.
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monensin 0.4mg monensin 04mg+ tiamulin 3.0mg
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This conclusion is supported by the parasitological data given in Tables 1 to 4 and Figure 1, showing the increased anticoccidial efficacy of the polyethers. The development of side effects such as depressed weight gain at high monensin levels can be considered as the consequence of an "overdosing" of monensin caused by its slower degradation. This assumption is supported by data from Ryley and Betts (1973) and Damron et al. (1977) who demonstrated a reduction in body weight with increasing monensin concentrations. In contrast, there was no disadvantageous effect of a combination of lasalocid and tiamulin even at the highest concentration used. Thus the increased tissue levels resulted in improved efficacy without toxicological side effects within the dose range tested. Finally, it is well known that an improvement of weight gain in chickens infected with E. tenella can be achieved by using antibiotics as feed additives (Ohe and Arakawa, 1975). The weight gain noted in these experiments for chickens treated with tiamulin alone may thus result in part from its activity against gram positive bacteria (Drews et al., 1975). However, this possibility is nonconfirmed and remains to be tested. REFERENCES
FIG. 2. Concentrations of monensin in plasma of the perfusion medium used in experiments with the isolated perfused rat liver.
Challey, J. R., and T. K. Jeffers, 1973. Synergism between 4-hydroxyquinoline and pyridone coccidiostats. J. Parasitol. 59:502-504.
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a
% in drinking water
TIAMULIN AND POLYETHER ANTIBIOTICS
tenella field isolates. Poultry Sci. 56:979-984. Mitzkat, H. J., 1968. Der Status des energieliefernden Stoffwechsels der Leber bei Diabetes mellitus. Habilitationsschrift, Medizinische Hochschule, Hannover. Ohe, O., and A. Arakawa, 1975. Effect of feed additive antibiotics on chickens infected with Eimeria tenella. Poultry Sci. 54:1008-1018. Perry, R. W., W. M. Beeson, and M. T. Mohler, 1976. Effect of monensin on beef cattle performance. J. Anim. Sci. 42:761-765. Raun, A. P., C. O. Cooley, E. L. Potter, R. P. Rathmacher, and L. F. Richardson, 1976. Effect of monensin on feed efficacy of feed lot cattle. J. Anim. Sci. 43:670-677. Ryley, J. F., 1975. Lerbek, a synergistic mixture of methyl-benzoquate and clopidol for the prevention of chicken coccidiosis. Parasitology 70:377—384. Ryley, J. F., and M. J. Betts, 1973. Chemotherapy of chicken coccidiosis. Adv. Pharmacol. Chemother. 11:221-293. Schimassek, H., 1963. Metabolites of carbohydrate metabolism in the isolated perfused rat liver. Biochem. Z. 336:460-467. Stipkovits, L., G. Laber, and E. Schiitze, 1978. Field trial tests new antibiotic. Pig Int. 8, 32:34—35. Weppelman, R. M., G. Olson, D. A. Smith, T. Tamas, and A. van Iderstine, 1977. Comparison of anticoccidial efficacy, resistance and tolerance o f narasin, monensin and lasalocid in chicken battery trials. Poultry Sci. 56:1550-1559. Westley, J. W., 1975. The polyether antibiotics: monocarboxylic acid ionophores. Annu. Rep. Med. Chem. 10:246-256.
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Damron, B. L., R. H. Harms, A. S. Arafa, and D. M. Janky, 1977. The effect of dietary lasalocid or monensin in the presence of roxarsone and graded methionine levels in broiler performance and processing characteristics. Poultry Sci. 56:1487— 1491. Drews, J., A. Georgopoulos, G. Laber, E. Schiitze, and J. Unger, 1975. Antimicrobial Activities of 81.723 hfu, a new pleuromutilin derivative. Antimicrobial Agents Chemother. 7:507-516. Golab, T., S. J. Barton, and R. E. Scroggs, 1973. Colorimetric method for monensin. JAOAC 56:171-173. Laber, G., and E. Schiitze, 1975. In vivo efficacy of 81.723 hfu, a new pleuromutilin derivative against experimentally induced airsacculitis in chicks and turkey poults. Antimicrobial Agents Chemother. 6:517-521. Meingassner, J. G., J. Hildebrandt, R. Leskova, and H. Mieth, 1977. Laboratory studies on the anticoccidial activity of septamycin. Poultry Sci. 56:1281-1288. Mitrovic, M., and E. G. Schildknecht, 1975a. Lasalocid: Resistance and cross-resistance studies in Eimeria tenella-infected chicks. Poultry Sci. 54:750—756. Mitrovic, M., and E. G. Schildknecht, 1975b. Comparative anticoccidial activity and compatibility of Lasalocid in broiler chickens. Poultry Sci. 54: 757-761. Mitrovic, M., E. G. Schildknecht, and C. Trainor, 1977. Effects of lasalocid and monensin in combination with roxarsone on lesion reduction and oocyst suppression in chicks infected with Eimeria
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1979 Poultry Sci 58:308-313 INTRODUCTION T h e anticoccidial activity of monensin stimulated such interest in polyether antibiotics t h a t m o r e t h a n 2 0 distinct c o m p o u n d s of this class have been reported during t h e last few years (Westley, 1 9 7 5 ; Weppelman etal., 1 9 7 7 ) . Several are n o w widely used alone or in combination with o t h e r drugs as p r o p h y l a c t i c agents against coccidiosis ( D a m r o n etal., 1 9 7 7 ; Mitrovic etal., 1 9 7 7 ; Mitrovic and Schildknecht, 1975a,b) or as feed additives in r u m i n a n t s (Perry et al, 1 9 7 6 ; Raun et al., 1 9 7 6 ) . Tiamulin (Tiamutin®) is a new pleuromutilin derivative (Drews et al., 1 9 7 5 ) soon t o be launched as a prophylactic and t h e r a p e u t i c drug against m y c o p l a s m a infections in p o u l t r y (Laber a n d Schiitze, 1 9 7 5 ) and pigs (Stipkovits etal., 1 9 7 8 ) . In the c o n t e x t of an evaluation of its compatibility with o t h e r drugs l a b o r a t o r y investigations were u n d e r t a k e n t o s t u d y t h e anticoccidial efficacy of m o n e n s i n and lasalocid in t h e presence of tiamulin. The present comm u n i c a t i o n reports t h e results of these experim e n t s and indicates t h a t although tiamulin alone is inactive, its c o m b i n a t i o n with b o t h polyether antibiotics enhances their anticoccidial activity. These findings were postulated t o result from a metabolic interaction b e t w e e n these c o m p o u n d s in t h e host. In order t o evaluate t h e t y p e of interaction, e x p e r i m e n t s were carried o u t using isolated, perfused rat
liver t o s t u d y t h e metabolic fate of monensin in t h e presence of tiamulin. MATERIALS AND METHODS Experiments in Chickens. Material and experimental procedures used in t h e present s t u d y have already been described (Meingassner et al, 1977). The e x p e r i m e n t s were c o n d u c t e d in batteries with day old broiler t y p e chickens inoculated with E. tenella at 4 days of age (day 0). The medicated feed was fed t w o days before infection (day —2) and c o n t i n u e d until t h e end of t h e e x p e r i m e n t s on day +7 (Experim e n t 1, 2, 4 ) or +8 ( E x p e r i m e n t 3). Chickens in E x p e r i m e n t 5, which was t e r m i n a t e d on day +14, were given n o n m e d i c a t e d feed from day +7 onwards. Tiamulin was administered via t h e drinking water for three days from t h e t i m e of infection. Measured parameters included mortality due t o coccidiosis, fecal score, lesion score, weight gain, and percentage weight gain (Experiments 1, 2, 3, 4) or oocyst o u t p u t ( E x p e r i m e n t 5) of t r e a t e d chicks in comparison t o infected, u n t r e a t e d or uninfected, u n t r e a t e d controls. F u r t h e r details of individual experiments and medication levels are given in t h e tables or figures. Biochemical studies. Rat Liver Perfusion. Livers of female Sprague Dawley rats (200 t o 2 5 0 g) were perfused using t h e m e t h o d s of Mitzkat ( 1 9 6 8 ) and Schimassek ( 1 9 6 3 ) with
308
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ABSTRACT The anticoccidial activities of monensin and lasalocid have been studied separately and in combination with tiamulin, a new pleuromutilin derivative. Combinations of constant tiamulin concentration (.0125%) in drinking water with various levels of polyether anticoccidials (6.3 to 125 ppm) in feed and conversely of constant levels of anticoccidials with various concentrations of tiamulin were used. The prophylactic efficacy of these combined treatments in battery raised broiler chickens infected with Eimeria tenella was evaluated. Assessment of the parameters mortality, weight gain, dropping scores, lesion scores, and oocyst output showed that simultaneous application of tiamulin significandy improved the anticoccidial activity of the polyethers. As tiamulin alone is without anticoccidial activity, this phenomenon was considered to result from an interaction between tiamulin and the polyethers leading to a slower metabolic degradation of the latter. Thus tissue levels adequate for maximum anticoccidial activity would be attained with lower polyether dose levels. Experiments using isolated perfused rat liver showed that elimination of monensin was reduced by 60% in the presence of tiamulin.
TIAMULIN AND POLYETHER ANTIBIOTICS
RESULTS AND DISCUSSION
Parasitological Data. Table 1 summarizes Experiment 1 in which the effect of monensin or tiamulin alone was compared with their efficacy in combination. Monensin at 125 ppm prevented mortality and largely suppressed clinical signs and the appearance of lesions in comparison to untreated controls. Tiamulin at .0125% in the drinking water (a dose calculated to provide 25 to 30 mg/kg body weight) exhibited no anticoccidial activity, and treated animals were as severely affected as the infected controls. When monensin at levels of 25 ppm to 125 ppm was combined with .0125%
tiamulin, a reduction in fecal score and lesion score was observed compared to 125 ppm monensin alone. Improved responses were observed using 12.5 ppm monensin plus .0125% tiamulin. Feeding monensin levels higher than 50 ppm in combination with .0125% tiamulin depressed body weight. However, lower monensin levels caused a marked improvement in weight gain over that achieved with 125 ppm monensin alone. The effect of monensin and tiamulin alone or in combination on oocyst production is shown in Figure 1. Tiamulin (.0125%) or monensin (12.5 ppm) alone exhibited no marked suppressive effect. However, a combination of both drugs at these concentrations resulted in a reduction of oocyst output to a level lower than that achieved with 125 ppm monensin alone. The anticoccidial activity of lasalocid in combination with a constant tiamulin concentration was studied in Experiment 2. The data are presented in Table 2 and show that lasalocid at 12.5, 25, 50, or 75 ppm plus tiamulin at .0125% resulted in an increase in body weight over the treatment with 75 ppm lasalocid alone. Groups medicated with 25 ppm lasalocid plus .0125% tiamulin exhibited improved fecal score, survival rate, lesion score, and weight gain when compared with lasalocid alone. Weight gain was not decreased by the addition of tiamulin to levels of 75 ppm lasalocid. The anticoccidial activity of the polyethers in combination with reduced tiamulin levels was studied in Experiments 3 and 4. A combination of 125 ppm monensin plus .00063 to .0100% tiamulin resulted in better coccidial control, as indicated by reduced fecal score and lesion score, than either drug alone (Table 3). However, a combination of high concentrations of both drugs depressed body weight as observed in Experiment 1. Better results were achieved when tiamulin was used at concentrations below .0025% in the combined treatments. Recommended levels of lasalocid plus various tiamulin concentrations gave excellent coccidiosis control in terms of weight gain, survival rate, and fecal and lesion score in comparison to lasalocid alone (Table 4). Indeed even 75 ppm lasalocid alone did not completely prevent coccidiosis, although it did control weight gain. However, some groups receiving combined therapy showed as much as 15% weight gain above that achieved with 75 ppm lasalocid alone.
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180 ml of medium (Tyrode, 2.6% bovine albumine and bovine erythrocytes with total of 10 g% hemoglobin). Thirty minutes after starting the perfusion, tritiated tiamulin (3.7 juCi/mg) and/or monensin (.4, 1, or 2 mg dissolved in pure dimethylsulfoxide (dest. in vacuo)) were added. Samples of the medium (2 to 4 ml) were taken at specified times during the 3 hr perfusion. The excreted bile was collected for determination of radioactivity and for thin layer chromatography. Thin Layer Chromatography (TLC). Medium samples were centrifuged and the proteins of the supernatant collected by recentrifugation after precipitation with 20 times the volume of A-grade methanol. This extraction of the precipitate was repeated twice. The methanol-phase was evaporated, the residue dissolved in methanol and chromatographed on TLC plates (Merck No. 5737). The plates were developed and sprayed according to Golab et al. (1973). Bile was chromatographed without any previous preparation. The densitometric determination of monensin was carried out with a Schoeffel spectrodensitometer at 515 nm. The amounts of monensin in the samples were determined by means of appropriate standards on each plate. The radioactivity of the samples was determined in a Packard Tri-Carb liquid scintillation counter M 3395. Drugs. Monensin, provided by Messrs. Kwizda, Vienna, was used in pure form for in vitro studies and/or as premix (Elancoban) in chickens. Lasalocid was obtained in pure form (lot No. 1804-114) from Hoffman-La Roche, Basle. A soluble powder of tiamulin (45%, lot No. KRV 310-H/HA2) was used for administration via the drinking water.
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MEINGASSNER ET AL.
TABLE 1. Anticoccidial efficacy of monensin and tiamulin separately or in combination at constant tiamulin concentration against E. tenella in the chick up to 7 days after infection3-*0^ (Experiment 1) Ppm in feed
Drugs Monensin Tiamulin Monensin + Tiamulin Monensin + Tiamulin Monensin + Tiamulin Monensin + Tiamulin Monensin + Tiamulin Monensin + Tiamulin Infected controls Non-infected controls
%in drinking water
125
+ + + + + 12.5 +
125 100 75 50 25
.0125 .0125 .0125 .0125 .0125 .0125 .0125
FS
S%
LS
WG
WG%
3.5 6.0 0 0 0 0 1 1 6.5 0
100 92 100 100 100 100 100 100 67 100
.9 2.7 0 0 .25 0 1.0 1.7 2.6 0
78.6 79.7 54.9 62.1 73.2 81.0 83.7 86.9 75.2 91.8
85.6 86.8 59.8 67.6 79.7 88.2 91.1 94.7 81.9 100.0
FS: Fecal score, S%: Survival rate; LS: Lesion score; WG: Weight gain; WG%: Percentage weight gain. b 2 X 6 chicks per group, inoculum: 5.104 oocysts, strain Hoe. Treatment: Monensin in feed from day —2 to +7, tiamulin in drinking water from day 0 to +3.
Tiamulin alone has no anticoccidial activity. Thus its in vivo augmentation of the efficacy of the polyethers is not comparable with the synergistic effects demonstrated between 4hydroxyquinoline and pyridine coccidiostats (Challey and Jeffers, 1973) or clopidol and methylbenzoquate (Ryley, 1975). Neither does tiamulin alone, or in combination with polyethers, act indirectly by an inhibition of the
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^i^ •* 1
* •
monensin 125 ppm monensin 12.5 ppm tiamulin 0.0125 1. tiamulin 0.0125 "'• monensin 12.5 ppm infected controls
10 6
10 5 -
10 4 -
//— 6
7 8 9 10 11 Days after inoculation
12
13
14
FIG. 1. The effect of monensin and tiamulin separately or in combination on the oocyst output of chickens infected with E. tenella (Experiment 5). 2 X 6 chicks per group, inoculum: 5.103 oocysts, strain Hoe. Treatment: monensin in feed (—2 -> 7 days), tiamulin in drinking water (0 -* 3 days).
excystation process (measured by intact oocysts recovery from the feces in the 24 hr following inoculation) or by alteration of gut mobility (Meingassner, unpublished data). Thus it is probable that the enhancement of the anticoccidial activity of the polyether antibiotics by tiamulin results from some interactions with the hosts' metabolism. Biochemical data. As a direct test of this proposition, the metabolic fate of monensin and tiamulin separately or in combination were monitored using isolated, perfused rat liver (IPL). Addition of 2 mg of monensin to IPL markedly altered its functions. Temporary swelling occurred in conjunction with a lower perfusion rate, while an elevation of potassium levels in the perfusion medium indicated cell damage. The rate of urea production decreased by 50% over a 2 hr period pointing to a lowering of gluconeogenesis, and, indeed, glucose levels did not rise any more. In addition, very low levels of lactate and extremely high levels of pyruvate occurred in the perfusion medium indicating a disturbance in liver redox potential. Less pronounced, but still observable signs of liver disturbance (e.g. the rate of urea production was reduced for 1 hr) were produced by 1 mg of monensin. However, .4 mg of monensin only reduced urea production for 30 min. It was thus considered to be a tolerable dose for the liver and proved sufficient for the study of the kinetics of monensin elimination from the perfusion medium. The polyether was elimi-
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a
TIAMULIN AND POLYETHER ANTIBIOTICS
311
TABLE 2. Anticoccidial efficacy of lasalocid and tiamulin separately or in combination at constant tiamulin concentration against E. tenella in the chick up to 7 days after infection2-*®'>c (Experiment 2)
Drugs Lasalocid Tiamulin Lasalocid + Tiamulin Lasalocid + Tiamulin Lasalocid + Tiamulin Lasalocid + Tiamulin Lasalocid + Tiamulin Infected controls Non-infected controls
Ppm in feed
% in drinking water
75
.0125 75 +.0125 50 +.0125 25 +.0125 12.5+ .0125 6.3 + .0125
FS
S%
LS
WG
WG%
3.5 8.5 0 0
96 63 100 100 100 92 54 45 100
1.5 2.8 0 .15 1.1 2.8 3.0 2.3 0
86.1 67.7 95.9 93.2 94.9 88.4 77.5 61.0 86.0
100.1 78.7 111.5 108.4 110.4 102.8 90.1 70.9
1.75 4.8 7.5 8.0 0
100
4 X 6 chicks per group, inoculum: 2.10 s oocysts, strain Hoe. Treatment: Lasalocid in feed from day —2 to +7, tiamulin in drinking water from day 0 to +3.
nated via t h e bile at an exponential rate with a half life of 7 min (Figure 2 ) . However, t h e various m e t a b o l i t e s h a d different colors a n d T L C properties, t h u s making quantitative analysis impossible. Up t o 10 mg of H 3 tiamulin was well tolerated b y t h e liver with n o n e of t h e above signs of i m p a i r m e n t . App r o x i m a t e l y 4 0 % of a 3 mg dose was m e t a b olized within 2.5 hr and excreted via t h e bile. When 3 mg of tiamulin was given s i m u l taneously with .4 mg of monensin t h e elimina-
tion rate of t h e l a t t e r was reduced resulting in a half life of 17 min. T h e excretion of m e t a b olites via t h e bile also proceeded m o r e slowly. Under these conditions t h e signs of incompatibility observed (elevation of potassium, high lactate, and p y r u v a t e levels in t h e perfusion m e d i u m and a lower rate of urea p r o d u c tion for 75 min) were c o m p a r a b l e with those for 1 mg of monensin alone. During this period t h e monensin c o n c e n t r a t i o n in t h e perfusion m e d i u m exceeded t h a t in e x p e r i m e n t s
TABLE 3. Anticoccidial efficacy of monensin and tiamulin separately or in combination at constant monensin concentration against E. tenella in the chick up to 8 days after infection3-*"*'1 (Experiment 3)
Drugs Monensin Tiamulin Monensin + Tiamulin Monensin + Tiamulin Monensin + Tiamulin Monensin + Tiamulin Monensin + Tiamulin Monensin + Tiamulin Monensin + Tiamulin Infected controls Non-infected controls
Ppm in feed
% in drinking water
125
125 125 125 125 125 125 125
.0125 + .0100 +.0075 + .0050 + .0025 + .00125 + .00063 + .00031
FS
S%
LS
WG
WG%
4.3 8.0 0 0 1.7 1.7 3.0 2.7 4.0 6.0 0
100 94 100 100 100 100 100 100 94 94 100
1.3 2.2 0 .1 .5 .1 .7 .3 .7 1.9 0
102.5 79.2 81.1 80.1 86.1 89.4 104.9 109.9 109.0 81.3 109.4
93.7 72.4 74.1 73.2 78.7 81.7 95.9 100.4 99.6 74.3 100.0
FS: Fecal score, S%: Survival rate; LS: Lesion score; WG: Weight gain; WG%: Percentage weight gain. 3 X 6 chicks per group, inoculum: 6.10 4 oocysts, strain Hoe. c
Treatment: Monensin in feed from day - 2 to +8, tiamulin in drinking water from day 0 to +3.
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FS: Fecal score, S%: Survival rate; LS: Lesion score; WG: Weight gain; WG%: Percentage weight gain.
MEINGASSNER ET AL.
312
TABLE 4. Anticoccidial efficacy oflasalocid and tiamulin separately or in combination at constant lasalocid concentration against E. tenella in the chick up to 7 days after infection3-!0'1- (Experiment 4) Ppm in feed
Drugs Lasalocid Tiamulin Lasalocid + Tiamulin Lasalocid + Tiamulin Lasalocid + Tiamulin Lasalocid + Tiamulin Lasalocid + Tiamulin Infected controls Non-infected controls
75 75 75 75 75 75
.0125 + .0075 + .0050 + .0025 + .00125 + .00063
FS
S%
LS
WG
WG%
3.5 8.5 0 0 0 .3 .7 8.0 0
96 63 100 100 100 100 100 45 100
1.5 2.8 0 0 .1 .1 .2 2.3 0
86.1 67.7 96.4 99.9 99.0 99.6 93.8 61.0 86.0
100.1 78.7 112.1 116.2 115.1 115.8 109.1 70.9 100
FS: Fecal score, S%: Survival rate; LS: Lesion score; WG: Weight gain; WG%: Percentage weight gain. 4 X 6 chicks per group, inoculum: 2.10 s oocysts, strain Hoe. Treatment: Lasalocid in feed from day —2 to +7, tiamulin in drinking water from day 0 to +3.
without tiamulin. However, monensin had no effect on the rate of tiamulin elimination. The metabolites found in the plasma were identical with those found without monensin, only the quantities excreted differed slightly. The experiments indicate that simultaneous administration of tiamulin and monensin induces a slower degradation and elimination of the latter from isolated, perfused rat liver. If this response also occurs in chickens, it is likely that the high efficacy of relatively low monensin levels also results from its slower elimination, thus producing tissue levels adequate for maximum anticoccidial activity.
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This conclusion is supported by the parasitological data given in Tables 1 to 4 and Figure 1, showing the increased anticoccidial efficacy of the polyethers. The development of side effects such as depressed weight gain at high monensin levels can be considered as the consequence of an "overdosing" of monensin caused by its slower degradation. This assumption is supported by data from Ryley and Betts (1973) and Damron et al. (1977) who demonstrated a reduction in body weight with increasing monensin concentrations. In contrast, there was no disadvantageous effect of a combination of lasalocid and tiamulin even at the highest concentration used. Thus the increased tissue levels resulted in improved efficacy without toxicological side effects within the dose range tested. Finally, it is well known that an improvement of weight gain in chickens infected with E. tenella can be achieved by using antibiotics as feed additives (Ohe and Arakawa, 1975). The weight gain noted in these experiments for chickens treated with tiamulin alone may thus result in part from its activity against gram positive bacteria (Drews et al., 1975). However, this possibility is nonconfirmed and remains to be tested. REFERENCES
FIG. 2. Concentrations of monensin in plasma of the perfusion medium used in experiments with the isolated perfused rat liver.
Challey, J. R., and T. K. Jeffers, 1973. Synergism between 4-hydroxyquinoline and pyridone coccidiostats. J. Parasitol. 59:502-504.
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% in drinking water
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