Anticoccidial Efficacy of Narasin in Battery Cage Trials

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1 Anticoccidial Efficacy of Narasin in Battery Cage Trials T. K. JEFFERS, L. V. TONKINSON, and M. E. CALLENDER Lilly Research Laboratories, Division of Eli Lilly and Company, P.O. Box 708, Greenfield, Indiana (Received for publication August 26, 1987) ABSTRACT Narasin is a polyether monocarboxylic acid antibiotic produced by Streptomyces aureofaciens. An extensive series of battery cage trials was conducted to evaluate the efficacy of narasin against recent field isolates of the pathogenic species of chicken coccidia. Statistical analyses of the results of these studies revealed that each successive increase in the concentration of narasin produced a significant reduction in the severity of cecal and intestinal lesions when compared with those in infected nonmedicated controls. Increasing the concentration of narasin also produced significant improvements in weight gain and feed efficiency when compared to gain and feed efficiency of infected nonmedicated controls. Maximum weight gain of birds infected with Eimeria tenella alone was obtained at a narasin concentration of 60 ppm, but birds infected with E. tenella plus intestinal species of coccidia, when medicated with 80 ppm narasin had weight gains significantly greater than those of birds medicated with 60 ppm narasin. Weight gain improvement decreased at narasin concentrations &100 ppm. Results of these studies confirm the effectiveness of narasin in controlling coccidial infections produced by recent field isolates of the pathogenic species of chicken coccidia. (Key words: narasin, anticoccidial efficacy, coccidiosis, Eimeria, ionophorous antibiotic) INTRODUCTION Narasin (Elanco Products Co., Indianapolis, IN) is a polyether monocarboxylic acid fermentation product of Streptomyces aureofaciens characterized by Boeck et al. (1977) and Berg and Hamill (1978). The ion selectivity spectrum of narasin is similar to that of monensin (Elanco Products Co., Indianapolis, IN) in that both have a high affinity for monovalent cations (Wong et al., 1977). The anticoccidial activity of narasin against three pathogenic species of chicken coccidia (Eimeria acervulina, E. maxima, and E. tenella) was first reported by Weppelman et al. (1977). In a series of three battery trials, Ruff et al. (1979) confirmed the efficacy of narasin against these three species as well as against three additional pathogenic species of coccidia, E. mivati (= E. mitis), E. necatrix, and E. brunetti. In order to more fully establish the relationship between narasin concentrations and anticoccidial effectiveness, an extensive series of battery cage trials was conducted in which narasin-medicated chickens were infected with five pathogenic species of coccidia (E. acervulina, E. brunetti, E. maxima, E. necatrix, and E. tenella) in single and mixed-species infections. The results of these studies are reported here Poultry Science 67: MATERIALS AND METHODS Animals. Coccidia-free broiler-type cockerels (Hubbard) were used routinely in these experiments. At 8 days of age, birds were pen-balanced by weight in groups of five birds/pen in wire-floored battery cages. Different groups of birds were placed on a standard broiler starter ration containing a final concentration of 0, 40, 60, 80, 100, or 120 ppm narasin. Several pen replicates were included in each treatment and the appropriate infected nonmedicated and noninfected nonmedicated controls were included in each experiment. Coccidial Infections. Birds were orally inoculated with sporulated oocysts of coccidia 48 h after initiation of medication. Ninety-nine coccidia isolates were used in the series of battery trials. Although some of these isolates were laboratory strains originally isolated before the advent of the polyether ionophore anticoccidials, the majority of the isolates were obtained from field origin samples, either from litter or intestinal samplings. Although various oocyst numbers were used in the inocula for different experiments depending upon the species composition of the inoculum, the intent in each experiment was to produce clinical coccidiosis in the infected nonmedicated birds. 1043

2 1044 JEFFERS ET AL. Experimental Parameters. Most experiments were terminated on the 6th or 7th day postinoculation, at which time the maximum expression of clinical signs of infection would be expected to be present in the infected nonmedicated birds. Mean weight gains were calculated as the mean weight gain per survivor and gain:feed ratio data were collected by dividing the total weight gain per cage by the total feed consumed per cage during the experimental period. Gaimfeed ratios were calculated only for cages in which there was no mortality, thus eliminating the necessity for the introduction of adjustments for mortality during the test period. Postmortem macroscopic examination of coccidial lesions was conducted on each bird and the severity of lesions was scored (0 to 4) as described by Johnson and Reid (1970). The severity of lesions due to monospecific coccidial infections was scored for the area of the intestine most affected by the particular species, whereas severity of lesions due to multiple-species infections was scored in each of three intestinal areas (duodenum, jejunum, and ileum) and cecal pouches. All areas were then added together to produce a total lesion score (0 to 16) that could be expressed as the total lesion score or further subdivided to be expressed as the total intestinal lesion score (0 to 12), or the average lesion score per lesion-scoring area (0 to 4). Statistical Analyses. Results of 104 of the trials were amenable to overall statistical analyses in which each trial was treated as one randomized block in the overall design. Trials were divided into separate sets for statistical analysis based upon the species of coccidia present in the cultures used as the inoculum in respective trials, as shown in Table 1. The inoculum in 65 of the 104 trials consisted of Eimeria tenella alone (37 trials) or E. tenella plus intestinal species (E. acervulina or E. maxima or both) of coccidia (28 trials). The overall results of these 65 trials were thought to characterize the anticoccidial efficacy of narasin against coccidia species typifying those present in commercial broiler production houses in the US (Jeffers, 1974). The inoculum in the remaining 39 trials consisted of one or more of the intestinal species of coccidia. These were doseresponse trials and each set was analyzed by linear plateau techniques (Anderson and Nelson, 1975). The variables analyzed were lesion scores, weight gain, and feed efficiency. All lesion score data were analyzed from the log transformation. The preferred form of feed efficiency data was gaimfeed ratio, as with this form the residual variation was more normally distributed than with the usual form of feed:gain ratio. Data and results of the statistical analyses are presented in dose-response graphs. The points in the figures are least squares means and if the line connecting two points is not horizontal, the difference between the points is statistically significant (P=.05). However, if the line is horizontal or flat between or among points, then the points are not significantly different from each other. RESULTS AND DISCUSSION Infections Produced by Eimeria tenella Alone. Cecal lesion scores for narasin-medicated birds infected either by laboratory strains or field isolates of E. tenella were significantly reduced by each increase in narasin concentration (Figure 1). There was a significant (P<.0001) difference in lesion score between that of infected nonmedi- TABLE 1. Summary of the total number of battery trials and number of pen replicates used in the evaluation of the anticoccidial efficacy of narasin against five pathogenic species of chicken coccidia Species composition of inoculum Battery trials Pen replicates in each treatment Eimeria tenella Eimeria tenella plus intestinal species Eimeria acervulina Eimeria brunetti Eimeria maxima Eimeria necatrix Mixed intestinal species Total

3 NARASIN IN BATTERY CAGE TRIALS *fc E3--- Field isolates Lab strains EL "a- ""El TOO 120 FIGURE 1. Response of cecal lesion scores to various levels of narasin when infection was produced by inoculation with Eimeria tenella alone. Where lines between or among points are not horizontal, differences between levels of narasin are significant (P<.05). The difference between lab strains and field isolates was significant (P<.05). cated birds and that of infected narasin-medicated birds. The field isolates of E. tenella produced significantly more severe cecal lesions than did the laboratory strains. Weight gain of narasin-medicated birds was significantly (P<.0001) greater than that of infected nonmedicated birds (Figure 2). Although increases in narasin concentration up to 60 ppm significantly increased weight gain, there was no difference between gains of the 60, 80, and 100-ppm treatments in birds infected with field isolates. However, there was a significant decrease in the weight gain of birds medicated with narasin at concentrations of 100 or 120 ppm and infected with laboratory strains of E. tenella. This reduction in weight gain was drug related and was not observed in birds infected with field isolates of E. tenella, because the effects of the more severe coccidial infection on the weight gain of these birds was counterbalanced by increased efficacy of greater concentrations of narasin. These joint effects tended to mask the drug-related effect on weight gain that was evident only in the less severely infected birds. Feed efficiency of infected narasin-medicated groups was significantly improved when com * - ^ - Field isolates --{H3 Lab strains OO 120 FIGURE 2. Response of weight gain and gain:feed ratio to various levels of narasin when infection was produced by inoculation with Eimeria tenella alone. Where lines between or among points are not horizontal, differences between levels of narasin are significant (P<.05).

4 1046 JEFFERS ET AL. pared with that of the infected nonmedicated controls (Figure 2). Increasing concentrations of narasin up to 60 ppm significantly improved feed efficiency of birds infected with field isolates of E. tenella, whereas concentrations up to 80 ppm significantly improved the feed efficiency of birds infected with laboratory strains of E. tenella. Infections Produced by Eimeria tenella in Combination with Eimeria acervulina or Eimeria maxima or Both. Medication with narasin significantly reduced the severity of intestinal lesions in birds infected with E. tenella in combination with E. acervulina or E. maxima or both (Figure 3). Mixed species containing field strains of E. tenella produced more severe lesions than those containing laboratory strains. Each increase in the concentration of narasin produced a significant decrease in intestinal lesions produced by field isolates of coccidia. Concentrations ^40 ppm almost completely suppressed intestinal lesions in birds infected with laboratory strains of coccidia. All concentrations of narasin reduced the severity of cecal lesions in birds infected with either field strains or laboratory strains of E. tenella in combination with E. acervulina or E. maxima or both (Figure 3). Field isolates Lab strains At all concentrations, narasin improved weight gain over that of the nonmedicated infected controls (Figure 4). Weight gain increased with each increase in the concentration of narasin up to 80 ppm for birds infected with field isolates of E. tenella in combination with E. acervulina or E. maxima or both. Only 40 ppm narasin was needed for maximum weight gain of birds infected with laboratory strains of coccidia. Infected narasin-medicated groups also had significantly improved feed efficiency when compared with that of infected nonmedicated controls. Although significant improvement in feed efficiency occurred with increasing concentrations of narasin up to 60 ppm for birds infected with field isolates, there was no additional response with increasing concentrations of narasin up to 120 ppm. Birds medicated with narasin concentrations from 40 to 120 ppm and infected with laboratory strains of E. tenella in combination with the intestinal species E. acervulina or E. maxima or both had equivalent feed efficiencies. Infections Produced by Intestinal Species of Eimeria. All levels of dietary narasin reduced lesion severity (Figure 5). Although each concentration of narasin was equally effective in reducing the severity of lesions due to infection El- ' 2- --El- Field isolates Lab strains OO 120 FIGURE 3. Response of intestinal and cecal lesion scores to various levels of narasin when infection was produced by inoculation with Eimeria tenella plus intestinal species of coccidia. Where lines between or among points are not horizontal, differences between levels of narasin are significant (P<.05).

5 NARASIN IN BATTERY CAGE TRIALS 1047 ioo- 1 OO i Field isolates f 3 Lab strains SO TOO 1 20 FIGURE 4. Response of weight gain and gain:feed ratio to various levels of narasin when infection was produced by inoculation with Eimeria tenella plus intestinal species of coccidia. Where lines between or among points are not horizontal, differences between levels of narasin are significant (P<.05). 4- s+j - E. acervulina -{E3 E. brxinetti. -0 E. -maxima E. necatrix *fc Mixed 1 OO 1 20 FIGURE 5. Response of intestinal lesion scores to various levels of narasin when infection was produced by inoculation with four separate species and a mixed group of intestinal coccidia. Where lines between or among points are not horizontal, differences between levels of narasin are significant (P<.05); E. = Eimeria. with E. acervulina or E. necatrix, there was a linear reduction of lesion scores with all increasing concentrations of narasin in birds infected with E. brunetti or mixed intestinal species and for increasing concentrations up to 60 ppm for E. maxima. Narasin significantly improved the weight gain and feed efficiency of birds infected with each of the intestinal species of coccidia singly or in mixtures (Figure 6). Feed Assays. Assays of the narasin concentration in rations used in this series of battery cage trials are presented in Table 2. Concentrations of narasin by assay did not differ greatly from the respective theoretical drug concentrations. The only previous reports on the anticoccidial efficacy of narasin in battery cage trials are those of Weppelman et al. (1977) and Ruff et al. (1979). Weppelman et al. (1977) compared the activity of narasin, monensin, and lasalocid against two field strains of E. acervulina, five field strains of E. maxima, and three field strains of E. tenella. They reported that the efficacy of narasin against the different coccidial strains was parallel to that of monensin. Unfortunately, assays of the anticoccidial drug concentrations in the rations used in their studies were not ob-

6 1048 JEFFERS ET AL " 200- p-j- -4D 0.65" 3* 140~ CD 120' TOO' Ifi '. etc e-ruazz ina -Q E. br-u.rt.etti. 0 E. m-a.3cvm.cl E. neca.t'ricc fc Mixed 1OO " it) - E. cccer-vulvrlct. -Q E. brxunetti. -^ E. ma.a:vma. E. necatricc ** Mixed FIGURE 6. Response of weight gain and gain:feed ratio to various levels of narasin when infection was produced by inoculation with four separate species and a mixed group of intestinal coccidia. Where lines between or among points are not horizontal, differences between levels of narasin are significant (P<.05); E. = Eimeria. tained, thus making it difficult to precisely correlate drug concentrations and anticoccidial activity. The studies of Ruff et al. (1979) were more extensive in that narasin was tested against six species of coccidia (E. acervulina, E. brunetti, E. maxima, E. mivati, E. necatrix, and E. tenella). Narasin at concentrations of 60, 80, or 100 ppm significantly improved weight gain and feed conversion ratios when compared to those of infected nonmedicated controls. The maximum improvement in these parameters was obtained by feeding 80 ppm narasin. Lesion severity was also reduced in narasin-medicated birds. However, narasin assays were also omitted for the rations used in these studies, again preventing definitive assessment of associations between narasin concentrations and the degree of anticoccidial efficacy. Despite these deficiencies in the determination of narasin concentrations by assay, the results of these workers are in general agreement with the overall results of the present more extensive studies in clearly demonstrating the anticoccidial efficacy of narasin against pathogenic species of chicken coccidia. Although laboratory strains of coccidia were more readily controlled than were field strains, a 60 to 80-ppm TABLE 2. Summary of feed assay results for rations used in 104 battery cage evaluations of the anticoccidial efficacy of narasin against five pathogenic species of chicken coccidia Theoretical concentration of narasin Samples assayed Assay result (X ± SEM) (ppm) (no.) (ppm) 41.6 ± ± ± ± 2.52

7 NARASIN IN BATTERY CAGE TRIALS 1049 range of narasin concentrations was highly effective against coccidial infections as measured by reductions in the severity of each of the infections. REFERENCES Anderson, R. L., and L. A. Nelson, A family of models involving intersecting straight lines and concomitant experimental designs useful in evaluating response to fertilizer nutrients. Biometrics 31: Berg, D. H., and R. L. Hamill, The isolation and characterization of narasin, a new polyether antibiotic. J. Antibiot. (Tokyo) 31:1-6. Boeck, L. D., M. M. Hoehn, R. E. Kastner, R. W. Wetzel, N. E. Davis, and J. E. Westhead, a new polyether antibiotic: discovery and fermentation studies. Dev. Ind. Microbiol. 18:471^85. Jeffers, T. K., Eimeria acervuuna and E. maxima: Incidence and anticoccidial drug resistance of isolants in major broiler-producing areas. Avian Dis. 18: Johnson, J., and W. M. Reid, Anticoccidial drugs: Lesion scoring technique in battery and floor-pen experiments with chickens. Exp. Parasitol. 28: Ruff, M. D., W. M. Reid, J. K. Johnson, and W. A. Anderson, Anticoccidial activity of narasin in battery-raised broilers. Poultry Sci. 58: Weppelman, R. M., G. Olson, D. A. Smith, T. Tamas, and A. Vanlderstine, Comparison of anticoccidial efficacy, resistance and tolerance of narasin, monensin and lasalocid in chicken battery trials. Poultry Sci. 56: Wong, D. T., D. H. Berg, R. H. Hamill, and J. R. Wilkinson, Ionophorous properties of narasin, a new polyether monocarboxylic acid antibiotic, in rat liver mitochondria. Biochem. Pharmacol. 26: