The effect of combining subtherapeutic concentrations of different ionophorous antibiotics on anticoccidial action in chickens

J. COMP.

PATH.

1981. VOL.

THE EFFECT CONCENTRATIONS ANTIBIOTICS

91.

503

OF

COMBINING SUBTHERAPEUTIC OF DIFFERENT IONOPHOROUS ON ANTICOCCIDIAL ACTION CHICKENS

T. E. MCQUISTION* Department of Poultry

andL.

IN

R. MCDOUGALD~ of Georgia,

Science, Uniuersity Athens, Georgia 30602, U.S.A.

INTRODUCTION

Coccidiosis in poultry is controlled by preventive medication with a drug mixed into the feed. Continuous medication during the growing period is usually successful in avoiding diseaseloss, provided that the drugs are effective against the species of coccidia present on the farm, and that drug resistance has not become a problem. Anticoccidial drugs are sometimes combined in an effort to achieve a wider control of coccidia species or for synergistic effects. The combination of ethopabate and amprolium (Amprol Hi-E@), for instance, protects chickens against more speciesof coccidia than amprolium alone. The best known example of synergism is a combination of a sulfonamide drug with a 2,4-diaminopyrimidine, which produces synergism by blocking two separate biochemical reactions in the synthesis of tetrahydrofolate (Wang, Stotish and Poe, 1975; Rogers, Clark, Becker, Pessolono, Leanga, McManus, Audriuli and Cuckler, 1964; Ryley, 1973). More recently, methyl benzoquate and clopidol have been sold as a combined product (Lerbeka) because of synergistic effects of these compounds on coccidia (Ryley, 1975). The polyether ionophorous antibiotic compounds, monensin, lasalocid and salinomycin, are currently the most widely used anticoccidial drugs. The anticoccidial ionophores have affinity for monovalent and some divalent cations and cause translocation of these ions across cell membranes (Pressman, 1973, 1976). These compounds presumably react selectively with the intracellular coccidia causing a breakdown in membrane integrity and loss of homeostasis of the parasite. The differences in ion selectivity of the various anticoccidial ionophores suggest slightly different modes of action of the various drugs, raising the possibility of synergistic efficacy if the drugs are combined. Conversely, if the mode of action of these compounds is similar, then combination of subprophylactic dosesof 2 drugs might be additive in effectiveness against coccidia. This study was done to evaluate combinations of one-half the recommended prophylactic doses of monensin, lasalocid and salinomycin for control of Eimeria aceruulinaand E. tenella infections in young chickens and to determine whether synergistic or additive effects could be obtained. * Present address: t Correspondence 002 l-9975/81/040503

Department of Biology, to L. R. McDougald. +07

$02.00/O

Millikin

University,

Decatur,

Q 1981 Academic

Illinois Press Inc.

62522, (London)

U.S.A. Limited

504

T.

E.

MCQUISTION

MATERIALS

AND

AND

L.

R.

MCDOUGALD

METHODS

Animal experiments. Birds were obtained from a commercial hatchery as day-old broiler-type cockerel chicks (Cobb hybrid) and maintained in parasite-free accommodation with non-medicated starter ration (UGA starter mash) and water ad libitum. At 2 weeks of age, the birds were randomly divided into groups of 7 birds with nearly equal group body weights. The groups of birds were housed in steam sterilized, wire floor batteries with continuous lighting and the room temperature maintained at 26 “C. The birds were started on medicated feed when placed in the batteries. Commercial drug premixes of monensin* (Coban 45@), lasalocidt (Avatee@) and salinomycin$ (Coccistac@) were used to mix standard chick starter rations with the appropriate concentrations of drugs. Recently sporulated oocysts of E. tenella or E. acervulina (Sterwin strains) were given per OS at doses of 5.0 x lo4 or l-6 x lo6 per bird, respectively. Each experiment consisted of 11 treatments, with 3 replicate groups per treatment. The treatments were monensin, lasalocid or salinomycin mixed in the feed at the concentration recommended by the manufacturer (full dose; 100, 75 or 60 ppm, respectively). The same drugs were mixed into feed at half the recommended concentration (half dose), and 2 of the drugs were mixed in combination at half dose. In a separate experiment, the drugs were tested in a factorially arranged dose titration at 50 per cent, 34 per cent or 17 per cent of recommended doses. Non-medicated-infected and nonmedicated-uninfected control groups were included in each experiment. Medication was started 2 days prior to inoculation of oocysts and was continued until day 7 post-inoculation (PI). On day 7 PI, the birds were killed and the coccidial lesions in the intestine were ranked on a scale of 0 to +4 (Johnson and Reid, 1970). Feed consumption and weight gains were recorded for each group of birds. The data were evaluated by analysis of variance and the Duncan’s (1955) multiple range tests. In vitro exfieriments. Culture of E. tenella in chick kidney cells and treatment with drugs was done as previously described (McDougald and Galloway, 1976). Cultures were infected with sporozoites of E. tenella, then dosed with monensin, lasalocid, or salinomycin (O*OOl, O-1 or O-01 pg per ml, respectively). Coverslip cultures were examined live under phase contrast microscopy after 24 h to determine the effect of the drug on the parasites, and representative parasites were photographed.

RESULTS

All 3 ionophorous antibiotics, when used in the feed at the recommended concentration (full dose), were about equally effective in controlling caecal lesions of E. tenella (Fig. 1). Treatment with a combination of 2 drugs at half dose was as effective in controlling caecal lesions as single drugs used at full dose. There were significantly (P -=cO-05) more severe lesions in the birds given single drugs at half dose than birds given 2 drugs in combination of half doses. However, the birds given single drugs at half dose had less severe lesions than the infected,

non-medicated

control

birds.

Similarly, with infections of E. acervulina, all drugs were effective in controlling intestinal lesions when given at full dose (Fig. 2). When two drugs were used in combination at half dose, the birds had as few lesions as those infected birds given one drug at full dose. Individual drugs given at half dose were significantly * Eli Lilly & Co., Indianopolis, U.S.A. t Hoffman La Roche, Inc., Nutley, U.S.A. $ Chas. Pfizer, Inc., Groton, U.S.A.

COMBINATIONS

OF

ANTICOCCIDIAL

505

IONOPHORES

(P < 0.05) less effective than single drugs at full dose or combined drugs at half dose. The infected nonmedicated control birds had significantly higher lesion scores than any of the medicated birds.

I-

J

JNC IC

MO

LA

SA

MO

LA

SA

MO+SA

Fig.

LAtS MO+ LA

Treatments

1. Lesion scores of chickens infected with E. ten& and administered therapeutic (full dose) q subtherapeutic (half dose) 0 and combinations of subtherapeutic concentrations q of ionophorous antibiotics in the feed ration. MO, monensin; LA, lasalocid; SA, salinomycin; IC, infected control; NC, non-infected control.

LA

SA

MO

LA

SA

MOtSA

LAtSA MOtLA

Treatments

Fig.

2. Lesion scores of chickens infected with E. acervulina and administered therapeutic (full dose 0, subtherapeutic (half dose) 0 and combinations of subtherapeutic concentrations 0 of ionophorous antibiotics in the feed ration. MO, monensin; LA, lasalocid; SA, salinomycin; IC, infected control; NC, non-infected control.

506

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MCDOUGALD

The weight gains and feed conversion ratios generally correlated with the lesion scores (Table 1). The birds with monensin, 50 ppm, salinomycin, 30 ppm, or no medication in the feed ration had the highest feed conversion ratios (FCR) and lowest mean weight gains of the birds infected with E. tenella. All of the E. acervulina inoculated birds had similar feed conversion ratios except for a significantly higher FCR in the non-medicated infected group. The weight gains of most of the E. acervulina-infected birds were similar, except that birds given lasalocid (35 ppm), and the infected non-medicated controls had significantly lower weight gains. TABLE MEAN

WEIGHT

GAINS

AND FEED CONVERSIONS AND INFECTED WITH

Eimeria

GIVEN

arervulina

OR

IONOPHOROUS

(1) Infected control (2) Non-infected control (3) (4) (5)

Monensin, 100 ppm (full Lasalocid, 75 ppm Salinomycin, 60 ppm

dose)

(6) (7) (8)

Monensin, 50 ppm (half Lasalocid, 35 ppm Salinomycin, 30 ppm

dose)

(9)

Monensin, (combined Monensin, Lasalocid,

50 ppm + salinomycin, 30 ppm half doses) 50 ppm + lasalocid, 35 ppm 35 ppm + salinomycin, 30 ppm

ANTIBIOTIC

DRUGS

E. ten&a.

E. acervulina Weight Feed gain gain (54

Treatment

(10) (11)

1

OF CHICKENS

E. tenella Weight Feed gain pain

1482c 2090=

1 .882b 1.633&

2083& 20858 2105a

1.642& 1.560= 1.6258

2082a 1987b 2105a

I.6088 1.6568 1.625&

2097°C 2295= 1995c

1.958b 1.757&b 1.821&b

20758

1.643” 1.619& 1.565&

2362” 2327& 2422~

::Z=

Each value is the mean of 3 groups of 7 birds each. a,b,cJ Means with different superscripts in the same column

are statistically

13874 2362& 1.6788 f .2%b &

different

1.583:

(P < 0.05).

The factorial titration of drugs generally supported the results of the other studies and strengthened the hypothesis that the drugs were additive rather than synergistic (Table 2). When drugs were combined at one-half the recommended dose, the infection was controlled. Combinations of less than one-half the normal dose were less effective. The weight gains after challenge also supported the conclusion of additivity (Table 3). In Vitro Results The effect of the 3 drugs on intracellular sporozoites was similar to that previously reported for monensin (McDougald and Galloway, 1976) when observed at 24 h. Typically, sporozoites were vacuolated and misshapen, and the internal organelles were disorganized. Usually, the refractile body of the sporozoite was intact and of normal appearance. In most cultures a few of the sporozoites appeared normal, but very few parasites developed beyond the trophozoite stage.

COMBINATIONS

OF

ANTICOCCIDIAL

507

IONOPHORES

TABLE 2 LESION SCORES VARIOUS COMBINATIONS

GIVEN

Salinomycin concentration (ppm)

Monenri;

~ncentration

0

35

50

:i

3.00 2.50 1.57

2.29 1.29 1.71

1.50 0.86 1.29

30

1.29

1.71

1 .oo

0.71 Salinomyc;tbconcentr;$on 0

(ppm)

3.33 3.00 2,75 2.71

lesion groups

3.00 2.57 2.00 1.71

2.29 1.86 1.29 1.14

1.50 1.86 1.57 1.43

scores of groups of 7 birds. were not repeated but the data are listed

TABLE GAIN OF BIRDS INFECTED DRUGS IN A FACTORIAL

Monensi~ (ppm)

3.33 3.00 2.75 2.71 to complete

3.00 2.75 1.57 1.86

(ppm)

30 2.00 2.57 1.57 0.86

1.29 1.71 0.86 0.71

the table.

3

WITH COCCIDIA AND GIVEN VARIOUS COMBINATIONS OF IONOPHOROUS DESIGN (PER CENT GAIN AS COMPARED WITH CONTROLS) *

Salinomycin

concentration

DESIGN*?

(ppm)

3.33 3.00 2.00

24 36

WEIGHT

WITH COCCIDIA AND DRUGS IN A FACTORIAL

0

Lasalocid concentration

* Average t Control

OF BIRDS INFECTED OF IONOPHOROUS

T&a:?

(ppm)

50

0

65 18 2 94 Lasalocid coneen tra tion

94 92

88 85 Salinomycl;oconzentr;$on 0

( ppm)

0

65 2

:: 36 * Control

86 86

groups

89 were

not repeated,

93 zi

295

90 ii;

90

95

95

but the data

are listed

65 :: 89 to complete

(ppm)

30

:z 98

t:

i”;

95

:7

z;

the tables.

DISCUSSION

All paired combinations of monensin, lasalocid and salinomycin mixed in the diet at half the recommended prophylactic concentrations were effective in controlling E. acervulina and E. tenella infections in chickens. Although McDougald (1978a, b) described a synergistic action among combinations of several ionophorous compounds tested against E. tenella infections, the combinations in this study appeared to produce an additive effect in controlling intestinal and caecal coccidial lesions. The additive action of these compounds suggests a similar mode of action on the parasite. The anticoccidial action of these compounds is probably due to the physiological effects on transport of alkaline metal cations. Because of the diverse spectra of ionic specificity of various polyether ionophores (Pressman, 1976)) it is tempting to speculate that the mechanisms of anticoccidial action of these drugs may also be diverse. If this is the case, combinations of the drugs might

508

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MCQUISTION

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R.

MCDOUGALD

affect a broader range of ions and produce a synergistic effect. The fact that the anticoccidial effects were only additive, however, tends to refute this hypothesis, and suggests that the primary action is against the same physiologically important ion or group of ions. While many differences exist in the ion spectra of the drugs, all of the drugs have some affinity for sodium and potassium, and probably produce similar effects at the molecular level. Further, the similar effects of these drugs on the morphology of sporozoites of E. tenella in vitro support the suggestion of a similar mode of action of the 3 drugs. The effects of monensin, lasalocid and salinomycin observed in this experiment were consistent with previous observations on the action of monensin against coccidia in vitro (McDougald and Galloway, 1976). Further studies will be necessary to determine whether there is any practical application for the principle of combined doses of different ionophorous drugs. Such combinations would be of value if they resulted in : (a) savings in drug cost, (b) more effective control of parasites, or (c) less expression of side effects of the drugs used in the combination. Since no significant synergism was discovered, it is unlikely that (a) or (b) would result, but economic benefit would be realized if the combination allowed better weight gains or other aspects of performance. SUMMARY

The mode of action of polyether, ionophorous drugs against Eimeria infections in chickens was studied by feeding subeffective doses of drugs separately and in combination, in comparison with the full dose of each drug. Thus, monensin, lasalocid and salinomycin (50, 35 and 30 ppm, respectively) were compared with a full dose of the same drugs. The combinations of ionophorous antibiotics were as effective in controlling Eimeria tenella and E. acervulina infections as the single antibiotic mixed in the feed at recommended prophylactic concentrations. When intracellular sporozoites of E. tenella were treated with the drugs in vitro, the effects on morphology were similar, regardless of which drug was used. The additive effects of these drugs in the chicken, and their similar effects on intracellular sporozoites suggest that they have a similar mode of action on Eimeria sporozoites, despite differences in ion selectivity. REFERENCES

Duncan, D. B. (1955). Multiple range and multiple F tests. Biometrics, 11, l-42. 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. McDougald, L. R., and Galloway, R. B. (1976). Anticoccidial drugs: Effect on inactivity and survival intracellularly of Eimeria tenella sporozoites. Experimental Parasitolo.gy. 40; 3 14-3 19. McDougald, L. R. Tl978a). Coccidiocidal combinations. United States Patent No. 4.075.323. 21 Februarv 1978. McDougaid, L. R. (1978bj. Coccidiocidal combinations. United States Patent No. 4,083,962. 11 April 1978.

COMBINATIONS

OF

ANTICOCCIDIAL

IONOPHORES

509

Pressman, B. C. (1973). Properties of ionophores with broad range cation selectivity. Federation Proceedings, 32, 1698-l 703. Pressman, B. C. (1976). Biological applications of ionophores. Annual Review of Biochemistry, 45, 501-530. Rogers, E. F., Clark, R. L., Becker, H. J., Pessolono, A. A., Leanza, W. J., McManus, E. C., Andriuli, J. F., and Cuckler, A. C. (1964). Antiparasitic activity of 4amino-2-ethoxybenzoic acid and related compounds. Proceedings of the Society for Experimental Biology and Medicine, 117, 488-492. Ryley, J. F. (1973). Cytochemistry, physiology and biochemistry. In The Coccidia. D. M. Hammond and P. L. Long, Eds, University Park Press, Baltimore, pp. 145-181. Ryley, J. F. (1975). Lerbek, a synergistic mixture of methyl benzoquate and clopidol for the prevention of chicken coccidiosis. Parasitology, 70, 377-384. Wang, C. C., Stotish, R. L., and Poe, M. (1975). Dihydrofolate reductase from Eimeria tenella : Rationalization of chemotherapeutic efficacy of pyrimethamine. Journal of Protozoology, 22, 564-568. [Received for publication,

August 11 th, 19801