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Comparison of Anticoccidial Efficacy, Resistance and Tolerance of Narasin, Monensin and Lasalocid in Chicken Battery Trials
Comparison of Anticoccidial Efficacy, Resistance and Tolerance of Narasin, Monensin and Lasalocid in Chicken Battery Trials R. M. WEPPELMAN, G. OLSON, D. A. SMITH, T. TAMAS and A. VAN IDERSTINE Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 07065 (Received for publication January 20, 1977)
INTRODUCTION Anticoccidial activity has been claimed for many polyether antibiotics including monensin (Haney et al, 1970), lasalocid (Mitrovic and Schildknecht, 1973), nigericin (Gorman and Hamill, 1971), dianemycin (Hamill et al, 1969) and salinomycin (Kinashi et al, 1973; Itagaki et al., 1974), and the activities of monensin (Shumard and Callender, 1967; Reid, 1969; Reid et al, 1972; Biely, 1973; Callender et al., 1974;Clarices al, 1974) and lasalocid (Mitrovic, 1974; Mitrovic and Schildknecht, 1974, 1975; Mitrovic et al, 1975; Reid et al, 1975) have been the subjects of numerous publications. Monensin, the only polyether antibiotic to be marketed, has enjoyed considerable commercial success in spite of the reported development of resistance among Eimeria maxima strains (Jeffers, 1974). When fermentation broths were tested for anticoccidial activity in these laboratories, the polyether antibiotic narasin (Berg et al, 1976; Boeck et al, 1976; Wong, 1976) or Antibiotic A-28086, Factor A, patented by Eli Lilly (Berg et al, 1975), was rediscovered. The structure of this antibiotic, which is closely related to salinomycin (Kinashi et al, 1973) is presented in Figure 1, as are the structures of monensin and lasalocid. When narasin's identity was
established, it was decided to initiate a series of rigorously controlled tests to determine which characteristics it might share with lasalocid and monensin. The present report gives the results of these tests in which the anticoccidial efficacy, host tolerance, and projected resistance development of these three polyether antibiotics are compared.
MATERIALS AND METHODS Acquisition of Coccidial Cultures. The geographic origins of the cultures used in this study are presented in Table 1. The three strains with the LS prefix originally were obtained from the field but have been single cell isolated to assure strain purity and have been maintained in the laboratory for several years. The remaining strains were isolated from recent field outbreaks of coccidiosis. Diets. Laboratory Broiler Feed (Pennfield Corporation, Lancaster, Pa.), a complete diet free of drugs, served as basal ration for all experiments. The appropriate medications are added to this ration and the mixture was blended until uniformity resulted. Anticoccidial Efficacy Studies. The efficacy studies were conducted using two-week-old sex-separated Hubbard-Hubbard chickens ob-
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ABSTRACT The anticoccidial efficacy, host tolerance, and projected resistance development of the three polyether antibiotics, monensin, narasin, and lasalocid were compared. The efficacy of narasin against different coccidial strains was found to parallel that of monensin in as much as strains which were refractory to monensin were also refractory to narasin. In contrast, lasalocid easily controlled some strains which were not well controlled by either narasin or monensin and failed to control one strain readily controlled by these two antibiotics. In growing chicks, lasalocid at the projected use level of 75 p.p.m. and narasin at an efficacious level of 100 p.p.m. were both better tolerated than monensin at the recommended use level of 121 p.p.m. The frequency of mutants resistant to each of these polyether compounds was found to be less than 8.6 X 10" 9 per drug sensitive oocyst for one strain of Eimeria tenella. This corresponds to less than 0.036 and 0.148 as frequent as mutants of this strain resistant to glycarbylamide or to amquinate, respectively. Poultry Science 56:1550-1559, 1977
NARASIN, MONENSIN AND LASALOCID
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TABLE 1 .—Species and origin of coccidial strains Culture number
Species
Origin Lasolocid-A (Molecular Weight 636.9)
E. E. E. E. E. E. E. E. E. E.
acervulina acervulina tenella tenella tenella maxima maxima maxima maxima maxima
Indiana Maryland Mississippi Australia England Maryland Texas Alabama Maryland Texas
tained from a commercial h a t c h e r y . These chickens were individually weighed a n d those in t h e middle p o r t i o n of t h e weight distribution were r a n d o m l y assigned t o t h e e x p e r i m e n t a l pens o n an equal weight basis. Each t r e a t m e n t g r o u p consisted of replicate pens of five chickens ( o n e pen of males a n d o n e pen of females). T h e medicated diets were fed 4 8 h o u r s prior t o infection and were c o n t i n u e d for seven t o n i n e days post infection depending on t h e coccidial species. T h e coccidial infection was i n d u c e d b y administering a 1.0 ml. suspension of sporu-
CHI Mel
Me
Monensin (Molecular Weight 688.9
Me
Me
Et
Narosin (Molecular Weight 764.9)
FIG. 1. The structures of the three polyether antibiotics studied.
lated o o c y s t s containing either 5 0 , 0 0 0 Eimeria tenella, 1 0 0 , 0 0 0 Eimeria maxima or 2 0 0 , 0 0 0 Eimeria acervulina directly into t h e crop of each chicken with a b l u n t dosing needle at-
TABLE 2.—Comparative efficacy of narosin, monensin and lasalocid against E. acervulina strains Average oocysts per chicken X 10 6
Medication
Percent survival
Relative percent weight gain
Average lesion score
DP-681
None 60 p.p.m. narasin 80 p.p.m. narasin 100 p.p.m. narasin 75 p.p.m. monensin 100 p.p.m. monensin 125 p.p.m. monensin 75 p.p.m. lasalocid 100 p.p.m. lasalocid 125 p.p.m. lasalocid
100 100 100 100 100 100 100 100 100 100
45 110 110 87 98 105 104 110 110 112
3.2 0.6 0 0 1.2 1.0 0 0.8 0 0
16.9 1.6 0.6 0 2.8 2.4 0 4.7 0 0
DP-702
None 60 p.p.m. narasin 80 p.p.m. narasin 75 p.p.m. monensin 100 p.p.m. monensin 125 p.p.m. monensin 75 p.p.m. lasalocid 100 p.p.m. lasalocid 125 p.p.m. lasalocid
97 100 100 100 100 100 100 100 100
76 101 95 90 91 85 89 93 97
4.0 0.9 0 2.9
485 0.6 0.3 267 33 40 567.7 315.7 430.3
Merck strain number
1.3 1.1 3.3 2.7 1.9
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DP-681 DP-702 DP-628 DP-669 LS-18 DP-677 DP-700 LS-17 LS-47 FS-49
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R. M. WEPPELMAN ET AL. Chicken Growth Studies. Day-old sex-separated H u b b a r d - H u b b a r d chickens were o b t a i n e d from a commercial h a t c h e r y and reared in electrically-heated, wire floored, metal b a t t e r y b r o o d e r s in t e m p e r a t u r e controlled r o o m s . Water a n d feed were supplied w i t h o u t restriction. At t h r e e d a y s of age, t h e chickens in t h e middle one-third of t h e weight distribution were divided i n t o g r o u p s of either four or eight chickens equally balanced on an individual weight basis. T h e groups of chickens and t h e e x p e r i m e n t a l diets were separately assigned at r a n d o m t o t h e pen locations for each experim e n t . T h e e x p e r i m e n t a l diets were fed t o t h e chickens for 18 days c o m m e n c i n g when t h e chickens were 3 days old. T h e effect of t h e diet s u p p l e m e n t was evaluated in t e r m s of t h e relative weight gains of each g r o u p calculated as
TABLE $ .—Comparative efficacy ofnarasin, monensin and lasalocid against E. tenella strains
Merck strain number
Relative percent weight gain
Average lesion score
Average oocysts per chicken X 10 s
Medication
Percent survival
DP-628
None 60 p.p.m. narasin 80 p.p.m. narasin 100 p.p.m. narasin 75 p.p.m. monensin 100 p.p.m. monensin 125 p.p.m. monensin 75 p.p.m. lasalocid 100 p.p.m. lasalocid 125 p.p.m. lasalocid
70 100 100 100 100 100 100 100 100 100
53 89 92 87 79 81 81 99 97 93
3.6 1.2 1.0 0.9 1.8 l-.l 0.2 0.4 0.5 0.3
40 18 15 17 23 13 0.5 1.3 3.3 0.1
DP-669
None 60 p.p.m. narasin 80 p.p.m. narasin 100 p.p.m. narasin 75 p.p.m. monensin 100 p.p.m. monensin 125 p.p.m. monensin 75 p.p.m. lasalocid 100 p.p.m. lasalocid 125 p.p.m. lasalocid
69 100 100 100 100 100 93 100 100 100
41 95 97 83 98 93 73 106 99 104
3.8 0.1 0 0 0.1 0.5 0.3 0.3 0.1 0
45 1.4 0 0 2.3 6.0 0.5 1.0 0.4 0
LS-18
None 40 p.p.m. narasin 60 p.p.m. narasin 80 p.p.m. narasin 75 p.p.m. monensin 100 p.p.m. monensin 125 p.p.m. monensin 75 p.p.m. lasalocid 100 p.p.m. lasalocid 125 p.p.m. lasalocid
83 80 100 100 100 100 100 100 100 100
59 82 93 95 94 91 90 103 103 104
3.7 2.4 0.5 0.4 1.1 0.1 0 0.7 0.1 0
24.8 14.6 5.5 4.5 8.6 1.2 1.3 7.1 0.3 0
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tached t o a volumetric syringe. At t h e terminat i o n of t h e e x p e r i m e n t , t h e surviving chickens were weighed and sacrificed. T h e gross intestinal lesions were scored from 0 ( n o r m a l ) t o 4 . 0 ( m a x i m u m lesion or death d u e t o coccidiosis) as described b y McManus et aZ.(1968). Average oocyst o u t p u t per chicken was d e t e r m i n e d with h e m o c y t o m e t e r . Since, in every case, t h e t w o replicate pens yielded essentially identical results, t h e results f r o m t h e five pullets a n d t h o s e from t h e five cockerals have been pooled for presentation in Tables 2, 3 and 4 . F o r comparison purposes, t h e percent survival, t h e average weight gain relative t o uninfected, u n m e d i c a t e d controls, t h e average lesion score a n d t h e average oocyst o u t p u t per chicken have been c o m b i n e d i n t o an anticoccidial i n d e x (Table 5), as described b y McManus et al. ( 1 9 6 8 ) .
NARASIN, MONENSIN AND LASALOCID
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TABLE 4.—Comparative efficacy ofnarasin, monensin and lasalocid against E. maxima strains
Merck strain number
DP-700
LS-17
LS-47
FS-49
Average lesion score
Average oocysts per chicken X 106
None 6 0 p.p.m. 7 0 p.p.m. 8 0 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 125 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 125 p.p.m.
narasin narasin narasin monensin monensin monensin lasalocid lasalocid lasalocid
100 100 100 100 100 100 100 100 100 100
66 101 100 92 95 86 86 115 89 101
2.9 1.5 1.1 1.3 2.1 1.7 1.8 0 0 0
4.5 4.1 6.5 2.3 6.8 10.4 3.0 0 0 0
None 6 0 p.p.m. 8 0 p.p.m. 1 0 0 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 125 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 1 2 5 p.p.m.
narasin narasin narasin monensin monensin monensin lasalocid lasalocid lasalocid
100 100 100 100 100 100 100 100 100 100
65 101 103 98 102 95 75 84 102 98
2.8 0.6 0.7 0 1.0 0.7 0.4 0.1 0 0
3.3 2.5 0.3 0 1.7 0.6 0.3 0.3 0 0
None 6 0 p.p.m. 8 0 p.p.m. 1 0 0 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 125 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 125 p.p.m.
narasin narasin narasin monensin monensin monensin lasalocid lasalocid lasalocid
90 100 100 100 100 100 100 100 100 100
35 98 86 99 87 84 80 85 100 113
3.1 0.2 0.2 0.1 0.6 1.2 0.3 0 0 0
4.1 2.2 0 0.4 4.2 1.9 0.5 0 0 0
None 6 0 p.p.m. 8 0 p.p.m. 1 0 0 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 125 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 1 2 5 p.p.m.
narasin narasin narasin monensin monensin monensin lasalocid lasalocid lasalocid
100 100 100 100 100 100 100 100 100 100
83 100 101 91 99 94 89 102 98 100
2.7 0.1 0 0 0.1 0 0.2 0 0 0
3.9 0 0 0 1.3 0 1.2 0 0 0
None 6 0 p.p.m. 8 0 p.p.m. 1 0 0 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 125 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 125 p.p.m.
narasin narasin narasin monensin monensin monensin lasalocid lasalocid lasalocid
100 100 100 100 100 100 100 100 100 100
63 93 90 89 92 91 82 100 93 93
2.8 1.0 0.5 0 0.8 0.3 0 0 0.1 0
3.5 1.5 1.7 1.7 8.6 2.1 3.8 0 0 0
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DP-677
Medication
Percent survival
Relative percent weight gain
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R. M. WEPPELMAN ET AL.
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NARASIN, MONENSIN AND LASALOCID
RESULTS Anticoccidial Activities of Narasin, Monensin and Lasalocid. The activities of these antibiotics against strains of E. acervulina, E. tenella and E. maxima are shown in Tables 2, 3 and 4, respectively, and the anticoccidial indices resulting from these trials are tabulated in Table 5. Narasin at 60 p.p.m. produced excellent control of both E. acervulina strains and all E. tenella strains except DP-628 which was not well controlled by any level of narasin tested. This same strain was refractory to 75 p.p.m. and 100 p.p.m. monensin but was acceptably controlled by 125 p.p.m. monensin. E. acervulina strain DP-702 was controlled by the two highest levels of monensin but not by the lowest level, 75 p.p.m. The remaining E. acervulina strain, DP-681, and the remaining two E.
tenella strains, DP-669 and LS-18, were well controlled by 75 p.p.m. monensin. In contrast to these results, lasalocid at 75 p.p.m., the lowest level tested, achieved excellent control of all E. tenella strains and of E. acervulina strain DP-681. The remaining E. acervulina strain DP-702 was not adequately controlled by any level of lasalocid tested. All levels of lasalocid resulted in good control of the five strains of E. maxima tested. In contrast, strains DP-677 and FS-49 were not totally controlled by any level of either narasin or monensin. The remaining E. maxima strains, DP-700, LS-17 and LS-47, were controlled by at least the highest level of narasin or monensin. Table 6 presents the average anticoccidial indices resulting when each treatment was challenged with these ten strains, or in the case of 100 p.p.m. narasin, resulting from challenge with eight strains. The average indices from all three levels of narasin, monensin or lasalocid were different from no medication to at least the 0.05% level of significance for these strains. The Apparent Relationship Between Monensin and Narasin Sensitivity. The preceding data suggested that sensitivity to monensin and narasin might be correlated. For example, strains DP-677 and FS-49 were relatively refractory to both antibiotics while strains DP-669, LS-18, DP-681 and LS-47 were adequately controlled by even the lowest level of either medication (Table 5). Sensitivity to lasalocid on the other hand did not appear to be highly correlated with sensitivity to the other two coccidiostats. Lasalocid at all levels controlled all strains except DP-702, which was not controlled by even the highest level of this drug. In contrast, this strain was well controlled by narasin and by the upper two levels of monensin. To determine the relationships among the sensitivities to these three polyether compounds, the anticoccidal indices produced by a particular treatment and by an alternative treatment were paired by strain. The correlation coefficient between the two sets of indices was then calculated from the product moment formula. The significance level attached to the correlation coefficient was determined for a one tailed test from the T statistic where the degrees of freedom equalled two less than the number of paired indices used to calculate the coefficient. The results of this analysis are shown in Table 6. The indices resulting from 60 p.p.m.
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a percentage of the weight gain of the unmedicated control groups. Resistance Studies. The frequencies of E. tenella mutants resistant to narasin or to lasalocid were determined by the procedure previously used to determine the frequencies of mutants resistant to monensin and other anticoccidial drugs (Weppelman et ah, 1977). By this procedure, a preparation of sporulated oocysts of E. tenella strain LS-18, which is sensitive to both narasin and lasalocid, was divided into groups of 5 X 10 6 sporulated oocysts and each group was used to inoculate five chickens medicated with either 80 p.p.m. narasin or 75 p.p.m. lasalocid. Groups failing to reproduce during this passage were discarded and presumed to have contained no resistant mutants, while the progeny from those groups which did reproduce were subjected to a second passage through five chickens receiving the same medication. The progeny from those groups successfully reproducing during this second passage were subjected to a third sequential passage through medicated chickens. On the basis of oocyst production, lesion scores and fatalities during this third passage, it was decided whether any of the original groups had produced resistant descendants. The frequency of mutants in the original population used to inoculate the first passage was then calculated with the Poisson distribution from the fraction of the original groups which failed to produce resistant descendants and which by inference had contained no resistant mutants.
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R. M. WEPPELMAN ET AL.
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narasin correlated significantly with those from 80 p.p.m. narasin and those from all three levels of monensin but not with those from any lasalocid treatment. The indices from 80 p.p.m. narasin correlated significantly with those from 100 p.p.m. narasin and with those from all monensin treatments but not with the indices resulting from any lasalocid treatment. The indices from the monensin treatments correlated with each other but not with those from any lasalocid treatment. The indices resulting from the three lasalocid treatments correlated significantly only with each other. The correlation coefficients produced by comparing the anticoccidial indices resulting from treatment with 100 p.p.m. narasin with those resulting from the other treatments were variable. The indices from 100 p.p.m. narasin significantly correlated with those from 80 p.p.m. narasin and with those from 75 or 100 p.p.m. monensin but not with the indices from the remaining narasin or monensin treatments. Correlation with the indices from the three lasalocid treatments was either significantly positive, significantly negative, or positive but not significant. We feel that two factors account for these aberrant results. Only eight, rather than ten pairs of indices, were available for calculation of the correlation coefficient in as much as 100 p.p.m. narasin was not tested against two strains, LS-18 and DP-702 (Tables 2, 3 and 5). Secondly, the range of indices resulting from 100 p.p.m. narasin was narrower than the ranges resulting from the other treatments (Table 5) and it is difficult to develop meaningful correlations over such a narrow range. For these reasons, we feel the correlations observed with the two lower levels of narasin are more meaningful than those observed with 100 p.p.m. narasin.
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In summary, two types of correlation appear in Table 6. The indices resulting from one level of a drug tend to correlate with those from another level of the same drug, which is expected in as much as the response of a coccidial strain to a particular level of medication should be reflected in its response to another level of the same drug. The more noteworthy correlation was between the indices resulting from the narasin treatments and those resulting from the monensin treatments. This suggests that sensitivity, or resistance, to these two antibiotics goes hand in hand. In contrast, the response of the strains to lasalocid appeared unrelated to their response to either monensin
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NARASIN, MONENSIN AND LASALOCID
sage. The average number of sporulated oocysts produced by the seven narasin groups was 2 X 10 6 while that produced by the three lasalocid groups was 1.1 X 10 6 . When these seven narasin groups and three lasalocid groups were subjected to a third sequential passage through medicated chickens, none of the groups produced fatalities or dramatic caecal lesions. The numbers of oocysts yielded by the various groups were comparable to the numbers produced when medicated chickens were infected with wild type oocysts which had not been previously exposed to medication. It was concluded that none of the 24 groups, each comprised of the 5 X 10 6 sporulated oocysts used to inoculate the selection passage, had produced a clone of resistant descendants and by inference that none of these groups had contained a resistant mutant. Table 8 presents the maximum frequencies of mutants resistant to narasin or to lasalocid as calculated from the Poisson distribution. The frequencies of mutants resistant to monensin and to various other drugs, as presented previously (Weppelman et ah, 1977) are included for comparison.
DISCUSSION When chickens medicated with narasin, monensin, or lasalocid were challenged with dif-
TABLE 7.--Effect of narasin, monensin and Utsalocid on weight gains and ft•ed efficiency of broiler chickens from one -half to three weeks of age
Medication
No. chickens dead/ total
Weight gain (Relative percent)
Feed/gain (Relative percent)
None 10 p.p.m. 50 p.p.m. 100 p.p.m. 150 p.p.m.
narasin narasin narasin narasin
1/32 0/8 0/8 0/8 0/8
100 (346.2 g.) +2.9 +4.0 + 5.5 -16.6
100 (1.586 g./g.) -0.2 +1.4 -2.7 +4.7
None 60 p.p.m. 121 p.p.m. 171 p.p.m. 181 p.p.m.
monensin monensin monensin monensin
2/176 0/16 0/72 0/32 0/32
100 (360.7 g.) -1.9 -13.5 -32.2 -25.9
100 (1.575 g./g.) +1.2 -5.7 -12.0 -10.9
None 37.5 p.p.m. 75 p.p.m. 150 p.p.m. 300 p.p.m.
lasalocid lasalocid lasalocid lasalocid
2/32 0/16 2/16 0/16 2/16
100 (353.4 g.) +11.1 +3.4 -3.8 -33.1
100 (1.509 g./g.) -1.5 -2.5 +0.1 +8.7
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or to narasin. Tolerance. When narasin, monensin, or lasalocid was fed to chickens between one-half and three weeks of age in battery cases, weight gains and feed efficiency were as shown in Table 7. When judged on either feed efficiency or weight gain, narasin at levels of 100 p.p.m. or less and lasalocid at levels of 150 p.p.m. or less appear less toxic than monensin at the use level of 121 p.p.m., thus demonstrating that both narasin and lasalocid have greater therapeutic ratios than monensin. Frequency of Mutants Resistant to Narasin or to Lasalocid. When groups containing 5 X 10 6 sporulated E. tenella oocysts were each passed through five chickens medicated with either 80 p.p.m. narasin or 75 p.p.m. lasalocid, 18 of the 24 groups exposed to narasin and 22 of the 24 groups exposed to lasalocid produced detectable progeny (8 X 10 3 or more sporulated oocysts per group). The average number of oocysts produced by the 18 narasin groups was 1.7 X 10 6 and that produced by the 22 lasalocid groups was 4.1 X 10 6 . The 18 narasin groups and 22 lasalocid groups producing detectable sporulated oocysts during the first passage were subjected to a second sequential passage through five chickens receiving the same medication. Seven of the narasin groups and three of the lasalocid groups produced detectable progeny during this pas-
1557
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R. M. WEPPELMAN ET AL. TABLE 8.—Frequencies of mutants resistant to various coccidiostats
Avg. number of resistant mutants per group [ m ] '
Drugs having undetectable frequencies of resistance 80 p.p.m. narasin 75 p.p.m. lasalocid 121 p.p.m. monensin 3 125 p.p.m. nicarbazin 3 125 p.p.m. amprolium 3 3 5 p.p.m. cycostat 3
> > > > > >
< < < < < <
Drugs having detectable frequencies of resistance 60 p.p.m. glycarbylamide 3 30 p.p.m. amquinate 3
23/24 23/24 26/27 38/39 3 7/38 26/27
5/15 9/12
0.043 0.043 0.038 0.026 0.027 0.038
Frequency of resistant mutants 2
< < < < < <
1.2 0.28
8.6 X 10"' 8.6 X 10"' 7.5 X 10~9 5 X 10""9 5 X 10~9 7.5 X 10"'
2.4 X 10"7 5.8 X 10"8
'Calculated from the Poisson distribution as m = —In P(o). ^Calculated as m mutants per 5 X 106 sporulated oocysts from which each group was descended. 3 Data from Weppelman et al. (1977).
ferent coccidial strains, the response of a strain to narasin generally reflected its response to monensin (Table 6), suggesting that relative tolerance to monensin and relative tolerance to narasin occur collaterally. In contrast, the response of a strain to lasalocid appeared unrelated to its response to either narasin or monensin. These relationships might be explained by the different biochemical activities of these polyether antibiotics. All three are ionophores capable of transporting metal ions through biological membranes, but they differ in their ion selectivity. Narasin has essentially the same ion selectivity as its close chemical relative salinomycin (Burg, 1976), which optimally transports monovalent ions like Na + and K + but is ineffective in transporting divalent ions (Mitani et al, 1975). Monensin, as well as nigericin and dianemycin, has a similar ion selectivity (Pressman, 1968). Lasalocid, in contrast, is capable of transporting divalent metal ions like Mg + + and Ca + + in addition to monovalent ions (Pressman, 1973). This additional activity of lasalocid might be responsible for its ability to control such coccidial strains as DP-677 and FS-49 which were less sensitive to narasin and monensin (Tables 4 and 5). The unique biochemical activities of lasalocid might also account for the fact that it effected a better separation of anticoccidial
efficacy and toxicity than did either narasin or monensin (Table 7). Ryley and Wilson (1975) compared lasalocid with monensin and reached a similar conclusion. In turn, narasin at an efficacious level was found to be better tolerated than monensin at the use level of 121 p.p.m. (Table 7). It should however be noted that battery tolerance tests employing chickens between one-half to three weeks of age, as were performed here, tend to overstate the toxicity occurring under field conditions. In unpublished experiments we have found 121 p.p.m. monensin to be less toxic in battery tolerance tests when the experiment was extended to eight weeks rather than three weeks as was done in the experiment presented in Table 7. Furthermore, when an eight week growout was performed under simulated field conditions rather than in battery cages, 121 p.p.m. monensin appeared to have no demonstrable effect on either weight gain or feed efficiency. Evaluation of the frequencies of mutants resistant to monensin, lasalocid, or to narasin yielded values less than 8.6 X 10" 9 per wild type oocyst for all three drugs. Our failure to isolate a lasalocid-resistant mutant is consistent with the results of Mitrovic and Schildknecht (1975) who serially passed E, tenella 15 times through chickens medicated with either suboptimal (30 p.p.m.) or optimal levels (75 p.p.m.) of this drug and did not observe any
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Medication
Fractions of groups not yielding mutants [P(o)l
NARASIN, MONENSIN AND LASALOCID
REFERENCES Berg, D. H., R. L. Hamill and M. M. Hoehn, 1975. Antibiotic A-28086 and process for production thereof. U.S. Patent 7,506,920. Berg, D. H., R. L. Hamill and M. M. Hoehn, 1976. Narasin, a new polyether antibiotic produced by Streptomyces aureofaciens. Isolation, characterization and properties. Sixteenth Interscience Conf. Antimicrobial Agents Chemotherapy, Chicago, p. 235. Biely, J., 1973. Monensin in broiler rations. Avian Dis. 17:362-368. Boeck, L. D., M. M. Hoehn, R. E. Kastner, R. W. Wetzel, N. E. Davis and J. E. Westhead, 1976. Narasin, a new polyether antibiotic—discovery and fermentation studies. SIM News, 26:37—38. Burg, R. W., 1976. Merck Sharp & Dohme Research Laboratories. Unpublished observations. Callender, M. E., P. V. Lake and R. F. Shumard, 1974. Performance of monensin sodium in two premix forms in broilers in oocyst seeded litter. Proc. Abstr. 15th World Poultry Cong. Expo., New Orleans, pp. 9 9 - 1 0 1 . Clarke, M. L., M. Diaz, B. Guilloteau, P. L. Hudd and J. W. Stoker, 1974. European field evaluation of monensin, a new anticoccidial agent. Avian Pathol. 3:25-35. Gorman, M., and R. L, Hamill, 1971. Nigericin for treating coccidiosis. U.S. Patent 3,555,150. Hamill, R. L., M. M. Hoehn, G. E. Pittenger, J. Chamberlin and M. Gorman, 1969. Dianemycin, an antibiotic of the group affecting ion transport. J. Antibio. 22:161-164. Haney, M., M. Hoehn and J. McGuire, 1970. Novel Antibiotic A-3823. Complex and process for production thereof. U.S. Patent 3,501,568. Itagaki, K., M. Tsubokura and K. Otsuki, 1974.
Studies on methods for evaluation of anticoccidial drugs in vitro. Jap. J. Vet. Sci. 36:195-202. Jeffers, T. K., 1974. Eimeria acervulina and E. maxima: Incidence and anticoccidial drug resistance of isolants in major broiler-producing areas. Avian Dis. 18:331-342. Kinashi, H., N. Otake and H. Yonehara, 1973. The structure of salinomycin, a new member of the p o l y e t h e r antibiotics. Tetrahedron Letters, 49:4955-^1958. McManus, E. C , W. C. Campbell and A. C. Cuckler, 1968. Development of resistance to quinoline coccidiostats under field and laboratory conditions. J. Parasitol. 54:1190-1193. Mitani, M., T. Yamanishi and Y. Miyazaki, 1975. Salinomycin: A new monovalent cation ionophore. Biochem. Biophys. Res. Commun. 66:1231—1236. Mitrovic, M., 1974. Anticoccidial activity of lasalocid in chickens. Third Int. Cong. Parasitol., Munich, 3:1319-1320. Mitrovic, M. and E. G. Schildknecht, 1973. Anticoccidial activity of Antibiotic X-537A in chickens. Poultry Sci. 52:2065. Mitrovic, M. and E. G. Schildknecht, 1974. Anticoccidial activity of lasalocid (X-537A) in chicks. Poultry Sci. 53:1448-1455. Mitrovic, M., and E. G. Schildknecht, 1975. Lasalocid: Resistance and cross-resistance studies in Eimeria tenella-infected chicks. Poultry Sci. 54:750—756. Mitrovic, M., E. G. Schildknecht and W. L. Marusich, 1975. Comparative anticoccidial activity and compatibility of lasalocid in broiler chickens. Poultry Sci. 54:757-761. Pressman, B. C , 1968. lonophorous antibiotics as models for biological transport. Fed. Proc. 27:1283-1288. Pressman, B. C , 1973. Properties of ionophores with b r o a d range cation selectivity. Fed. Proc. 32:1698-1703. Reid, W. M., 1969. Efficacy studies on some new a n t i c o c c i d i a l d r u g s . A c t a . V e t . (Brno) 38:137-145. Reid, W. M., J. Johnson and J. Dick, 1975. Anticoccidial activity of lasalocid in control of moderate and severe coccidiosis. Avian Dis. 19:12—18. Reid, W. M., L. Kowalski and J. Rice, 1972. Anticoccidial activity of monensin in floor-pen experiments. Poultry Sci. 51:139-146. Ryley, J. F., and R. G. Wilson, 1975. Laboratory studies with some recent anticoccidials. Parasitol. 70:203-222. Shumard, R. F., and M, E. Callender, 1967. Monensin, a new biologically active compound VI. Anticoccidial a c t i v i t y . Antimicrob. Agents Chemothera.:369-377. Weppelman, R. M., J. A. Battaglia and C. C. Wang, 1977. Eimeria tenella: The selection and frequency of drug resistant mutants. Exp. Parasitol. In press. Wong, D. T., 1976. The ionophorous properties of narasin in rat liver mitochondria. Sixteenth Interscience Conf. Antimicrobial Agents Chemotherapy, Chicago, p. 236.
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decrease in sensitivity. Even though lasalocidresistant mutants appear to be quite rare and are not readily isolated in the laboratory, we have found a field strain, DP-702 (Table 2), which is tolerant to this drug. This strain, which presumably had never been exposed to lasalocid previously, appears to be an indigenous strain which is intrinsically tolerant to lasalocid rather than a true drug-resistant mutant derived from drug-sensitive parents. Although there have been no reports describing the development of monensin-resistant strains in the laboratory, Jeffers (1974) has reported the emergence of monensin-resistant E, maxima in the field. Our data also include some recent E. maxima field isolates which might be regarded as tolerant t o monensin (Tables 4 and 5).
1559
INTRODUCTION Anticoccidial activity has been claimed for many polyether antibiotics including monensin (Haney et al, 1970), lasalocid (Mitrovic and Schildknecht, 1973), nigericin (Gorman and Hamill, 1971), dianemycin (Hamill et al, 1969) and salinomycin (Kinashi et al, 1973; Itagaki et al., 1974), and the activities of monensin (Shumard and Callender, 1967; Reid, 1969; Reid et al, 1972; Biely, 1973; Callender et al., 1974;Clarices al, 1974) and lasalocid (Mitrovic, 1974; Mitrovic and Schildknecht, 1974, 1975; Mitrovic et al, 1975; Reid et al, 1975) have been the subjects of numerous publications. Monensin, the only polyether antibiotic to be marketed, has enjoyed considerable commercial success in spite of the reported development of resistance among Eimeria maxima strains (Jeffers, 1974). When fermentation broths were tested for anticoccidial activity in these laboratories, the polyether antibiotic narasin (Berg et al, 1976; Boeck et al, 1976; Wong, 1976) or Antibiotic A-28086, Factor A, patented by Eli Lilly (Berg et al, 1975), was rediscovered. The structure of this antibiotic, which is closely related to salinomycin (Kinashi et al, 1973) is presented in Figure 1, as are the structures of monensin and lasalocid. When narasin's identity was
established, it was decided to initiate a series of rigorously controlled tests to determine which characteristics it might share with lasalocid and monensin. The present report gives the results of these tests in which the anticoccidial efficacy, host tolerance, and projected resistance development of these three polyether antibiotics are compared.
MATERIALS AND METHODS Acquisition of Coccidial Cultures. The geographic origins of the cultures used in this study are presented in Table 1. The three strains with the LS prefix originally were obtained from the field but have been single cell isolated to assure strain purity and have been maintained in the laboratory for several years. The remaining strains were isolated from recent field outbreaks of coccidiosis. Diets. Laboratory Broiler Feed (Pennfield Corporation, Lancaster, Pa.), a complete diet free of drugs, served as basal ration for all experiments. The appropriate medications are added to this ration and the mixture was blended until uniformity resulted. Anticoccidial Efficacy Studies. The efficacy studies were conducted using two-week-old sex-separated Hubbard-Hubbard chickens ob-
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ABSTRACT The anticoccidial efficacy, host tolerance, and projected resistance development of the three polyether antibiotics, monensin, narasin, and lasalocid were compared. The efficacy of narasin against different coccidial strains was found to parallel that of monensin in as much as strains which were refractory to monensin were also refractory to narasin. In contrast, lasalocid easily controlled some strains which were not well controlled by either narasin or monensin and failed to control one strain readily controlled by these two antibiotics. In growing chicks, lasalocid at the projected use level of 75 p.p.m. and narasin at an efficacious level of 100 p.p.m. were both better tolerated than monensin at the recommended use level of 121 p.p.m. The frequency of mutants resistant to each of these polyether compounds was found to be less than 8.6 X 10" 9 per drug sensitive oocyst for one strain of Eimeria tenella. This corresponds to less than 0.036 and 0.148 as frequent as mutants of this strain resistant to glycarbylamide or to amquinate, respectively. Poultry Science 56:1550-1559, 1977
NARASIN, MONENSIN AND LASALOCID
1551
TABLE 1 .—Species and origin of coccidial strains Culture number
Species
Origin Lasolocid-A (Molecular Weight 636.9)
E. E. E. E. E. E. E. E. E. E.
acervulina acervulina tenella tenella tenella maxima maxima maxima maxima maxima
Indiana Maryland Mississippi Australia England Maryland Texas Alabama Maryland Texas
tained from a commercial h a t c h e r y . These chickens were individually weighed a n d those in t h e middle p o r t i o n of t h e weight distribution were r a n d o m l y assigned t o t h e e x p e r i m e n t a l pens o n an equal weight basis. Each t r e a t m e n t g r o u p consisted of replicate pens of five chickens ( o n e pen of males a n d o n e pen of females). T h e medicated diets were fed 4 8 h o u r s prior t o infection and were c o n t i n u e d for seven t o n i n e days post infection depending on t h e coccidial species. T h e coccidial infection was i n d u c e d b y administering a 1.0 ml. suspension of sporu-
CHI Mel
Me
Monensin (Molecular Weight 688.9
Me
Me
Et
Narosin (Molecular Weight 764.9)
FIG. 1. The structures of the three polyether antibiotics studied.
lated o o c y s t s containing either 5 0 , 0 0 0 Eimeria tenella, 1 0 0 , 0 0 0 Eimeria maxima or 2 0 0 , 0 0 0 Eimeria acervulina directly into t h e crop of each chicken with a b l u n t dosing needle at-
TABLE 2.—Comparative efficacy of narosin, monensin and lasalocid against E. acervulina strains Average oocysts per chicken X 10 6
Medication
Percent survival
Relative percent weight gain
Average lesion score
DP-681
None 60 p.p.m. narasin 80 p.p.m. narasin 100 p.p.m. narasin 75 p.p.m. monensin 100 p.p.m. monensin 125 p.p.m. monensin 75 p.p.m. lasalocid 100 p.p.m. lasalocid 125 p.p.m. lasalocid
100 100 100 100 100 100 100 100 100 100
45 110 110 87 98 105 104 110 110 112
3.2 0.6 0 0 1.2 1.0 0 0.8 0 0
16.9 1.6 0.6 0 2.8 2.4 0 4.7 0 0
DP-702
None 60 p.p.m. narasin 80 p.p.m. narasin 75 p.p.m. monensin 100 p.p.m. monensin 125 p.p.m. monensin 75 p.p.m. lasalocid 100 p.p.m. lasalocid 125 p.p.m. lasalocid
97 100 100 100 100 100 100 100 100
76 101 95 90 91 85 89 93 97
4.0 0.9 0 2.9
485 0.6 0.3 267 33 40 567.7 315.7 430.3
Merck strain number
1.3 1.1 3.3 2.7 1.9
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DP-681 DP-702 DP-628 DP-669 LS-18 DP-677 DP-700 LS-17 LS-47 FS-49
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R. M. WEPPELMAN ET AL. Chicken Growth Studies. Day-old sex-separated H u b b a r d - H u b b a r d chickens were o b t a i n e d from a commercial h a t c h e r y and reared in electrically-heated, wire floored, metal b a t t e r y b r o o d e r s in t e m p e r a t u r e controlled r o o m s . Water a n d feed were supplied w i t h o u t restriction. At t h r e e d a y s of age, t h e chickens in t h e middle one-third of t h e weight distribution were divided i n t o g r o u p s of either four or eight chickens equally balanced on an individual weight basis. T h e groups of chickens and t h e e x p e r i m e n t a l diets were separately assigned at r a n d o m t o t h e pen locations for each experim e n t . T h e e x p e r i m e n t a l diets were fed t o t h e chickens for 18 days c o m m e n c i n g when t h e chickens were 3 days old. T h e effect of t h e diet s u p p l e m e n t was evaluated in t e r m s of t h e relative weight gains of each g r o u p calculated as
TABLE $ .—Comparative efficacy ofnarasin, monensin and lasalocid against E. tenella strains
Merck strain number
Relative percent weight gain
Average lesion score
Average oocysts per chicken X 10 s
Medication
Percent survival
DP-628
None 60 p.p.m. narasin 80 p.p.m. narasin 100 p.p.m. narasin 75 p.p.m. monensin 100 p.p.m. monensin 125 p.p.m. monensin 75 p.p.m. lasalocid 100 p.p.m. lasalocid 125 p.p.m. lasalocid
70 100 100 100 100 100 100 100 100 100
53 89 92 87 79 81 81 99 97 93
3.6 1.2 1.0 0.9 1.8 l-.l 0.2 0.4 0.5 0.3
40 18 15 17 23 13 0.5 1.3 3.3 0.1
DP-669
None 60 p.p.m. narasin 80 p.p.m. narasin 100 p.p.m. narasin 75 p.p.m. monensin 100 p.p.m. monensin 125 p.p.m. monensin 75 p.p.m. lasalocid 100 p.p.m. lasalocid 125 p.p.m. lasalocid
69 100 100 100 100 100 93 100 100 100
41 95 97 83 98 93 73 106 99 104
3.8 0.1 0 0 0.1 0.5 0.3 0.3 0.1 0
45 1.4 0 0 2.3 6.0 0.5 1.0 0.4 0
LS-18
None 40 p.p.m. narasin 60 p.p.m. narasin 80 p.p.m. narasin 75 p.p.m. monensin 100 p.p.m. monensin 125 p.p.m. monensin 75 p.p.m. lasalocid 100 p.p.m. lasalocid 125 p.p.m. lasalocid
83 80 100 100 100 100 100 100 100 100
59 82 93 95 94 91 90 103 103 104
3.7 2.4 0.5 0.4 1.1 0.1 0 0.7 0.1 0
24.8 14.6 5.5 4.5 8.6 1.2 1.3 7.1 0.3 0
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tached t o a volumetric syringe. At t h e terminat i o n of t h e e x p e r i m e n t , t h e surviving chickens were weighed and sacrificed. T h e gross intestinal lesions were scored from 0 ( n o r m a l ) t o 4 . 0 ( m a x i m u m lesion or death d u e t o coccidiosis) as described b y McManus et aZ.(1968). Average oocyst o u t p u t per chicken was d e t e r m i n e d with h e m o c y t o m e t e r . Since, in every case, t h e t w o replicate pens yielded essentially identical results, t h e results f r o m t h e five pullets a n d t h o s e from t h e five cockerals have been pooled for presentation in Tables 2, 3 and 4 . F o r comparison purposes, t h e percent survival, t h e average weight gain relative t o uninfected, u n m e d i c a t e d controls, t h e average lesion score a n d t h e average oocyst o u t p u t per chicken have been c o m b i n e d i n t o an anticoccidial i n d e x (Table 5), as described b y McManus et al. ( 1 9 6 8 ) .
NARASIN, MONENSIN AND LASALOCID
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TABLE 4.—Comparative efficacy ofnarasin, monensin and lasalocid against E. maxima strains
Merck strain number
DP-700
LS-17
LS-47
FS-49
Average lesion score
Average oocysts per chicken X 106
None 6 0 p.p.m. 7 0 p.p.m. 8 0 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 125 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 125 p.p.m.
narasin narasin narasin monensin monensin monensin lasalocid lasalocid lasalocid
100 100 100 100 100 100 100 100 100 100
66 101 100 92 95 86 86 115 89 101
2.9 1.5 1.1 1.3 2.1 1.7 1.8 0 0 0
4.5 4.1 6.5 2.3 6.8 10.4 3.0 0 0 0
None 6 0 p.p.m. 8 0 p.p.m. 1 0 0 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 125 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 1 2 5 p.p.m.
narasin narasin narasin monensin monensin monensin lasalocid lasalocid lasalocid
100 100 100 100 100 100 100 100 100 100
65 101 103 98 102 95 75 84 102 98
2.8 0.6 0.7 0 1.0 0.7 0.4 0.1 0 0
3.3 2.5 0.3 0 1.7 0.6 0.3 0.3 0 0
None 6 0 p.p.m. 8 0 p.p.m. 1 0 0 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 125 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 125 p.p.m.
narasin narasin narasin monensin monensin monensin lasalocid lasalocid lasalocid
90 100 100 100 100 100 100 100 100 100
35 98 86 99 87 84 80 85 100 113
3.1 0.2 0.2 0.1 0.6 1.2 0.3 0 0 0
4.1 2.2 0 0.4 4.2 1.9 0.5 0 0 0
None 6 0 p.p.m. 8 0 p.p.m. 1 0 0 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 125 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 1 2 5 p.p.m.
narasin narasin narasin monensin monensin monensin lasalocid lasalocid lasalocid
100 100 100 100 100 100 100 100 100 100
83 100 101 91 99 94 89 102 98 100
2.7 0.1 0 0 0.1 0 0.2 0 0 0
3.9 0 0 0 1.3 0 1.2 0 0 0
None 6 0 p.p.m. 8 0 p.p.m. 1 0 0 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 125 p.p.m. 75 p.p.m. 1 0 0 p.p.m. 125 p.p.m.
narasin narasin narasin monensin monensin monensin lasalocid lasalocid lasalocid
100 100 100 100 100 100 100 100 100 100
63 93 90 89 92 91 82 100 93 93
2.8 1.0 0.5 0 0.8 0.3 0 0 0.1 0
3.5 1.5 1.7 1.7 8.6 2.1 3.8 0 0 0
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DP-677
Medication
Percent survival
Relative percent weight gain
1554
R. M. WEPPELMAN ET AL.
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NARASIN, MONENSIN AND LASALOCID
RESULTS Anticoccidial Activities of Narasin, Monensin and Lasalocid. The activities of these antibiotics against strains of E. acervulina, E. tenella and E. maxima are shown in Tables 2, 3 and 4, respectively, and the anticoccidial indices resulting from these trials are tabulated in Table 5. Narasin at 60 p.p.m. produced excellent control of both E. acervulina strains and all E. tenella strains except DP-628 which was not well controlled by any level of narasin tested. This same strain was refractory to 75 p.p.m. and 100 p.p.m. monensin but was acceptably controlled by 125 p.p.m. monensin. E. acervulina strain DP-702 was controlled by the two highest levels of monensin but not by the lowest level, 75 p.p.m. The remaining E. acervulina strain, DP-681, and the remaining two E.
tenella strains, DP-669 and LS-18, were well controlled by 75 p.p.m. monensin. In contrast to these results, lasalocid at 75 p.p.m., the lowest level tested, achieved excellent control of all E. tenella strains and of E. acervulina strain DP-681. The remaining E. acervulina strain DP-702 was not adequately controlled by any level of lasalocid tested. All levels of lasalocid resulted in good control of the five strains of E. maxima tested. In contrast, strains DP-677 and FS-49 were not totally controlled by any level of either narasin or monensin. The remaining E. maxima strains, DP-700, LS-17 and LS-47, were controlled by at least the highest level of narasin or monensin. Table 6 presents the average anticoccidial indices resulting when each treatment was challenged with these ten strains, or in the case of 100 p.p.m. narasin, resulting from challenge with eight strains. The average indices from all three levels of narasin, monensin or lasalocid were different from no medication to at least the 0.05% level of significance for these strains. The Apparent Relationship Between Monensin and Narasin Sensitivity. The preceding data suggested that sensitivity to monensin and narasin might be correlated. For example, strains DP-677 and FS-49 were relatively refractory to both antibiotics while strains DP-669, LS-18, DP-681 and LS-47 were adequately controlled by even the lowest level of either medication (Table 5). Sensitivity to lasalocid on the other hand did not appear to be highly correlated with sensitivity to the other two coccidiostats. Lasalocid at all levels controlled all strains except DP-702, which was not controlled by even the highest level of this drug. In contrast, this strain was well controlled by narasin and by the upper two levels of monensin. To determine the relationships among the sensitivities to these three polyether compounds, the anticoccidal indices produced by a particular treatment and by an alternative treatment were paired by strain. The correlation coefficient between the two sets of indices was then calculated from the product moment formula. The significance level attached to the correlation coefficient was determined for a one tailed test from the T statistic where the degrees of freedom equalled two less than the number of paired indices used to calculate the coefficient. The results of this analysis are shown in Table 6. The indices resulting from 60 p.p.m.
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a percentage of the weight gain of the unmedicated control groups. Resistance Studies. The frequencies of E. tenella mutants resistant to narasin or to lasalocid were determined by the procedure previously used to determine the frequencies of mutants resistant to monensin and other anticoccidial drugs (Weppelman et ah, 1977). By this procedure, a preparation of sporulated oocysts of E. tenella strain LS-18, which is sensitive to both narasin and lasalocid, was divided into groups of 5 X 10 6 sporulated oocysts and each group was used to inoculate five chickens medicated with either 80 p.p.m. narasin or 75 p.p.m. lasalocid. Groups failing to reproduce during this passage were discarded and presumed to have contained no resistant mutants, while the progeny from those groups which did reproduce were subjected to a second passage through five chickens receiving the same medication. The progeny from those groups successfully reproducing during this second passage were subjected to a third sequential passage through medicated chickens. On the basis of oocyst production, lesion scores and fatalities during this third passage, it was decided whether any of the original groups had produced resistant descendants. The frequency of mutants in the original population used to inoculate the first passage was then calculated with the Poisson distribution from the fraction of the original groups which failed to produce resistant descendants and which by inference had contained no resistant mutants.
1555
R. M. WEPPELMAN ET AL.
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narasin correlated significantly with those from 80 p.p.m. narasin and those from all three levels of monensin but not with those from any lasalocid treatment. The indices from 80 p.p.m. narasin correlated significantly with those from 100 p.p.m. narasin and with those from all monensin treatments but not with the indices resulting from any lasalocid treatment. The indices from the monensin treatments correlated with each other but not with those from any lasalocid treatment. The indices resulting from the three lasalocid treatments correlated significantly only with each other. The correlation coefficients produced by comparing the anticoccidial indices resulting from treatment with 100 p.p.m. narasin with those resulting from the other treatments were variable. The indices from 100 p.p.m. narasin significantly correlated with those from 80 p.p.m. narasin and with those from 75 or 100 p.p.m. monensin but not with the indices from the remaining narasin or monensin treatments. Correlation with the indices from the three lasalocid treatments was either significantly positive, significantly negative, or positive but not significant. We feel that two factors account for these aberrant results. Only eight, rather than ten pairs of indices, were available for calculation of the correlation coefficient in as much as 100 p.p.m. narasin was not tested against two strains, LS-18 and DP-702 (Tables 2, 3 and 5). Secondly, the range of indices resulting from 100 p.p.m. narasin was narrower than the ranges resulting from the other treatments (Table 5) and it is difficult to develop meaningful correlations over such a narrow range. For these reasons, we feel the correlations observed with the two lower levels of narasin are more meaningful than those observed with 100 p.p.m. narasin.
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In summary, two types of correlation appear in Table 6. The indices resulting from one level of a drug tend to correlate with those from another level of the same drug, which is expected in as much as the response of a coccidial strain to a particular level of medication should be reflected in its response to another level of the same drug. The more noteworthy correlation was between the indices resulting from the narasin treatments and those resulting from the monensin treatments. This suggests that sensitivity, or resistance, to these two antibiotics goes hand in hand. In contrast, the response of the strains to lasalocid appeared unrelated to their response to either monensin
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NARASIN, MONENSIN AND LASALOCID
sage. The average number of sporulated oocysts produced by the seven narasin groups was 2 X 10 6 while that produced by the three lasalocid groups was 1.1 X 10 6 . When these seven narasin groups and three lasalocid groups were subjected to a third sequential passage through medicated chickens, none of the groups produced fatalities or dramatic caecal lesions. The numbers of oocysts yielded by the various groups were comparable to the numbers produced when medicated chickens were infected with wild type oocysts which had not been previously exposed to medication. It was concluded that none of the 24 groups, each comprised of the 5 X 10 6 sporulated oocysts used to inoculate the selection passage, had produced a clone of resistant descendants and by inference that none of these groups had contained a resistant mutant. Table 8 presents the maximum frequencies of mutants resistant to narasin or to lasalocid as calculated from the Poisson distribution. The frequencies of mutants resistant to monensin and to various other drugs, as presented previously (Weppelman et ah, 1977) are included for comparison.
DISCUSSION When chickens medicated with narasin, monensin, or lasalocid were challenged with dif-
TABLE 7.--Effect of narasin, monensin and Utsalocid on weight gains and ft•ed efficiency of broiler chickens from one -half to three weeks of age
Medication
No. chickens dead/ total
Weight gain (Relative percent)
Feed/gain (Relative percent)
None 10 p.p.m. 50 p.p.m. 100 p.p.m. 150 p.p.m.
narasin narasin narasin narasin
1/32 0/8 0/8 0/8 0/8
100 (346.2 g.) +2.9 +4.0 + 5.5 -16.6
100 (1.586 g./g.) -0.2 +1.4 -2.7 +4.7
None 60 p.p.m. 121 p.p.m. 171 p.p.m. 181 p.p.m.
monensin monensin monensin monensin
2/176 0/16 0/72 0/32 0/32
100 (360.7 g.) -1.9 -13.5 -32.2 -25.9
100 (1.575 g./g.) +1.2 -5.7 -12.0 -10.9
None 37.5 p.p.m. 75 p.p.m. 150 p.p.m. 300 p.p.m.
lasalocid lasalocid lasalocid lasalocid
2/32 0/16 2/16 0/16 2/16
100 (353.4 g.) +11.1 +3.4 -3.8 -33.1
100 (1.509 g./g.) -1.5 -2.5 +0.1 +8.7
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or to narasin. Tolerance. When narasin, monensin, or lasalocid was fed to chickens between one-half and three weeks of age in battery cases, weight gains and feed efficiency were as shown in Table 7. When judged on either feed efficiency or weight gain, narasin at levels of 100 p.p.m. or less and lasalocid at levels of 150 p.p.m. or less appear less toxic than monensin at the use level of 121 p.p.m., thus demonstrating that both narasin and lasalocid have greater therapeutic ratios than monensin. Frequency of Mutants Resistant to Narasin or to Lasalocid. When groups containing 5 X 10 6 sporulated E. tenella oocysts were each passed through five chickens medicated with either 80 p.p.m. narasin or 75 p.p.m. lasalocid, 18 of the 24 groups exposed to narasin and 22 of the 24 groups exposed to lasalocid produced detectable progeny (8 X 10 3 or more sporulated oocysts per group). The average number of oocysts produced by the 18 narasin groups was 1.7 X 10 6 and that produced by the 22 lasalocid groups was 4.1 X 10 6 . The 18 narasin groups and 22 lasalocid groups producing detectable sporulated oocysts during the first passage were subjected to a second sequential passage through five chickens receiving the same medication. Seven of the narasin groups and three of the lasalocid groups produced detectable progeny during this pas-
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R. M. WEPPELMAN ET AL. TABLE 8.—Frequencies of mutants resistant to various coccidiostats
Avg. number of resistant mutants per group [ m ] '
Drugs having undetectable frequencies of resistance 80 p.p.m. narasin 75 p.p.m. lasalocid 121 p.p.m. monensin 3 125 p.p.m. nicarbazin 3 125 p.p.m. amprolium 3 3 5 p.p.m. cycostat 3
> > > > > >
< < < < < <
Drugs having detectable frequencies of resistance 60 p.p.m. glycarbylamide 3 30 p.p.m. amquinate 3
23/24 23/24 26/27 38/39 3 7/38 26/27
5/15 9/12
0.043 0.043 0.038 0.026 0.027 0.038
Frequency of resistant mutants 2
< < < < < <
1.2 0.28
8.6 X 10"' 8.6 X 10"' 7.5 X 10~9 5 X 10""9 5 X 10~9 7.5 X 10"'
2.4 X 10"7 5.8 X 10"8
'Calculated from the Poisson distribution as m = —In P(o). ^Calculated as m mutants per 5 X 106 sporulated oocysts from which each group was descended. 3 Data from Weppelman et al. (1977).
ferent coccidial strains, the response of a strain to narasin generally reflected its response to monensin (Table 6), suggesting that relative tolerance to monensin and relative tolerance to narasin occur collaterally. In contrast, the response of a strain to lasalocid appeared unrelated to its response to either narasin or monensin. These relationships might be explained by the different biochemical activities of these polyether antibiotics. All three are ionophores capable of transporting metal ions through biological membranes, but they differ in their ion selectivity. Narasin has essentially the same ion selectivity as its close chemical relative salinomycin (Burg, 1976), which optimally transports monovalent ions like Na + and K + but is ineffective in transporting divalent ions (Mitani et al, 1975). Monensin, as well as nigericin and dianemycin, has a similar ion selectivity (Pressman, 1968). Lasalocid, in contrast, is capable of transporting divalent metal ions like Mg + + and Ca + + in addition to monovalent ions (Pressman, 1973). This additional activity of lasalocid might be responsible for its ability to control such coccidial strains as DP-677 and FS-49 which were less sensitive to narasin and monensin (Tables 4 and 5). The unique biochemical activities of lasalocid might also account for the fact that it effected a better separation of anticoccidial
efficacy and toxicity than did either narasin or monensin (Table 7). Ryley and Wilson (1975) compared lasalocid with monensin and reached a similar conclusion. In turn, narasin at an efficacious level was found to be better tolerated than monensin at the use level of 121 p.p.m. (Table 7). It should however be noted that battery tolerance tests employing chickens between one-half to three weeks of age, as were performed here, tend to overstate the toxicity occurring under field conditions. In unpublished experiments we have found 121 p.p.m. monensin to be less toxic in battery tolerance tests when the experiment was extended to eight weeks rather than three weeks as was done in the experiment presented in Table 7. Furthermore, when an eight week growout was performed under simulated field conditions rather than in battery cages, 121 p.p.m. monensin appeared to have no demonstrable effect on either weight gain or feed efficiency. Evaluation of the frequencies of mutants resistant to monensin, lasalocid, or to narasin yielded values less than 8.6 X 10" 9 per wild type oocyst for all three drugs. Our failure to isolate a lasalocid-resistant mutant is consistent with the results of Mitrovic and Schildknecht (1975) who serially passed E, tenella 15 times through chickens medicated with either suboptimal (30 p.p.m.) or optimal levels (75 p.p.m.) of this drug and did not observe any
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Medication
Fractions of groups not yielding mutants [P(o)l
NARASIN, MONENSIN AND LASALOCID
REFERENCES Berg, D. H., R. L. Hamill and M. M. Hoehn, 1975. Antibiotic A-28086 and process for production thereof. U.S. Patent 7,506,920. Berg, D. H., R. L. Hamill and M. M. Hoehn, 1976. Narasin, a new polyether antibiotic produced by Streptomyces aureofaciens. Isolation, characterization and properties. Sixteenth Interscience Conf. Antimicrobial Agents Chemotherapy, Chicago, p. 235. Biely, J., 1973. Monensin in broiler rations. Avian Dis. 17:362-368. Boeck, L. D., M. M. Hoehn, R. E. Kastner, R. W. Wetzel, N. E. Davis and J. E. Westhead, 1976. Narasin, a new polyether antibiotic—discovery and fermentation studies. SIM News, 26:37—38. Burg, R. W., 1976. Merck Sharp & Dohme Research Laboratories. Unpublished observations. Callender, M. E., P. V. Lake and R. F. Shumard, 1974. Performance of monensin sodium in two premix forms in broilers in oocyst seeded litter. Proc. Abstr. 15th World Poultry Cong. Expo., New Orleans, pp. 9 9 - 1 0 1 . Clarke, M. L., M. Diaz, B. Guilloteau, P. L. Hudd and J. W. Stoker, 1974. European field evaluation of monensin, a new anticoccidial agent. Avian Pathol. 3:25-35. Gorman, M., and R. L, Hamill, 1971. Nigericin for treating coccidiosis. U.S. Patent 3,555,150. Hamill, R. L., M. M. Hoehn, G. E. Pittenger, J. Chamberlin and M. Gorman, 1969. Dianemycin, an antibiotic of the group affecting ion transport. J. Antibio. 22:161-164. Haney, M., M. Hoehn and J. McGuire, 1970. Novel Antibiotic A-3823. Complex and process for production thereof. U.S. Patent 3,501,568. Itagaki, K., M. Tsubokura and K. Otsuki, 1974.
Studies on methods for evaluation of anticoccidial drugs in vitro. Jap. J. Vet. Sci. 36:195-202. Jeffers, T. K., 1974. Eimeria acervulina and E. maxima: Incidence and anticoccidial drug resistance of isolants in major broiler-producing areas. Avian Dis. 18:331-342. Kinashi, H., N. Otake and H. Yonehara, 1973. The structure of salinomycin, a new member of the p o l y e t h e r antibiotics. Tetrahedron Letters, 49:4955-^1958. McManus, E. C , W. C. Campbell and A. C. Cuckler, 1968. Development of resistance to quinoline coccidiostats under field and laboratory conditions. J. Parasitol. 54:1190-1193. Mitani, M., T. Yamanishi and Y. Miyazaki, 1975. Salinomycin: A new monovalent cation ionophore. Biochem. Biophys. Res. Commun. 66:1231—1236. Mitrovic, M., 1974. Anticoccidial activity of lasalocid in chickens. Third Int. Cong. Parasitol., Munich, 3:1319-1320. Mitrovic, M. and E. G. Schildknecht, 1973. Anticoccidial activity of Antibiotic X-537A in chickens. Poultry Sci. 52:2065. Mitrovic, M. and E. G. Schildknecht, 1974. Anticoccidial activity of lasalocid (X-537A) in chicks. Poultry Sci. 53:1448-1455. Mitrovic, M., and E. G. Schildknecht, 1975. Lasalocid: Resistance and cross-resistance studies in Eimeria tenella-infected chicks. Poultry Sci. 54:750—756. Mitrovic, M., E. G. Schildknecht and W. L. Marusich, 1975. Comparative anticoccidial activity and compatibility of lasalocid in broiler chickens. Poultry Sci. 54:757-761. Pressman, B. C , 1968. lonophorous antibiotics as models for biological transport. Fed. Proc. 27:1283-1288. Pressman, B. C , 1973. Properties of ionophores with b r o a d range cation selectivity. Fed. Proc. 32:1698-1703. Reid, W. M., 1969. Efficacy studies on some new a n t i c o c c i d i a l d r u g s . A c t a . V e t . (Brno) 38:137-145. Reid, W. M., J. Johnson and J. Dick, 1975. Anticoccidial activity of lasalocid in control of moderate and severe coccidiosis. Avian Dis. 19:12—18. Reid, W. M., L. Kowalski and J. Rice, 1972. Anticoccidial activity of monensin in floor-pen experiments. Poultry Sci. 51:139-146. Ryley, J. F., and R. G. Wilson, 1975. Laboratory studies with some recent anticoccidials. Parasitol. 70:203-222. Shumard, R. F., and M, E. Callender, 1967. Monensin, a new biologically active compound VI. Anticoccidial a c t i v i t y . Antimicrob. Agents Chemothera.:369-377. Weppelman, R. M., J. A. Battaglia and C. C. Wang, 1977. Eimeria tenella: The selection and frequency of drug resistant mutants. Exp. Parasitol. In press. Wong, D. T., 1976. The ionophorous properties of narasin in rat liver mitochondria. Sixteenth Interscience Conf. Antimicrobial Agents Chemotherapy, Chicago, p. 236.
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decrease in sensitivity. Even though lasalocidresistant mutants appear to be quite rare and are not readily isolated in the laboratory, we have found a field strain, DP-702 (Table 2), which is tolerant to this drug. This strain, which presumably had never been exposed to lasalocid previously, appears to be an indigenous strain which is intrinsically tolerant to lasalocid rather than a true drug-resistant mutant derived from drug-sensitive parents. Although there have been no reports describing the development of monensin-resistant strains in the laboratory, Jeffers (1974) has reported the emergence of monensin-resistant E, maxima in the field. Our data also include some recent E. maxima field isolates which might be regarded as tolerant t o monensin (Tables 4 and 5).
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