Sensitivity of Field Isolates of Eimeria from Two Broiler Complexes to Anticoccidial Drugs in the Chicken1

Sensitivity of Field Isolates of Eimeria from Two Broiler Complexes to Anticoccidial Drugs in the Chicken1 H. D. CHAPMAN and A. B. HACKER Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas 72701

1994 Poultry Science 73:1404-1408

tance is often correlated with the extent to which drugs have been used in the past. Coccidiosis is an important disease of The objective of this study, therefore, was poultry caused by parasites of the genus to investigate whether farms within the Eimeria that is controlled by the inclusion same complex will share patterns of of anticoccidial drugs in the feed. The resistance. widespread use of drugs, however, has resulted in the development of drug MATERIALS AND METHODS resistance. An indication of the compounds most likely to be effective can be obtained by conducting sensitivity tests on Birds coccidia obtained from commercial farms, A total of 720 Ross x Ross broiler chicks but the tests are both time consuming and were used in the experiments. Chicks were expensive. obtained from a local hatchery and reared Farms within a given geographical area until 12 d of age in wire-floored cages that are frequently part of a production com- had been steam sterilized before use. For plex that is served by a common feed mill each trial, 360 chicks were randomly alloand in which the same anticoccidial drug cated to 45 clean cages (8 birds per cage) programs are employed. Because it is not when 12 d old and placed on medicated possible to test isolates from every farm feed. They were given free access to water within a complex, it is customary to and a starter mash formulated to meet the examine one or two farms. It has been nutritional needs of the broiler chicken shown that the occurrence of drug resis- (National Research Council, 1984). INTRODUCTION

Drugs Received for publication March 21, 1994. Accepted for publication May 16, 1994. 'Published with the approval of the Director, Arkansas Agricultural Experiment Station.

The following drugs were incorporated in the feed at the concentrations indicated for the duration of the experiments:

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ABSTRACT The spectrum of resistance to seven currently used anticoccidial drugs in isolates of Eimeria obtained from farms from two broiler complexes was examined. All isolates were resistant to monensin, salinomycin, and narasin. Lasalocid was more effective in controlling coccidiosis than the other ionophores, although most isolates were classified as resistant to the drug. The majority of isolates were sensitive or showed reduced sensitivity to robenidine, clopidol, and decoquinate. It was concluded that for drugs that have been used extensively (e.g., monensin and salinomycin), examination of isolates from one or two farms may give results applicable to the entire complex. For drugs that have been used infrequently, however (such as robenidine, clopidol, and decoquinate), examination of isolates from at least five farms would be desirable to establish the spectrum of drug sensitivity. {Key words: Eimeria, drug resistance, sensitivity, anticoccidial drugs, broiler)

ANTICOCCIDIAL DRUG SENSITIVITY

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clopidol (Coyden®, 125 ppm), decoquinate (Deccox®; 30 ppm),2 lasalocid (Avatec®; 100 ppm),3 monensin (Coban®; 121 ppm),4 narasin (Monteban®; 80 ppm),4 robenidine (Robenz®; 33 ppm),5 and salinomycin (Biocox®; 66 ppm).6

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were nine treatments. Treatments 1 to 7 comprised birds that were infected and given the seven anticoccidial drugs. Treatments 8 and 9 comprised the unmedicated infected (UMI) and uninfected (UMU) controls. Individual isolates were not replicated. Two days after birds were given mediMedication at Commercial Sites cated feed (Day 0), they were weighed and Isolates from two broiler complexes inoculated with 200,000 oocysts. Six days (designated A and B) were studied. At both later (Day 6) they were weighed once more. complexes the ionophorous antibiotics Droppings in the pans were scored on Days salinomycin and monensin had been used 5 and 6 postinoculation to provide a for many years. Narasin had been em- subjective measure of the severity of infecployed for a single flock, but lasalocid had tion as described by Jeffers (1974). not been used. Clopidol had been used on two occasions Evaluation of Resistance recently at Complex A, but decoquinate and robenidine had not been employed for at The degree of resistance to each drug for least 10 yr. At Complex B, robenidine had each isolate was assessed by calculation of been used on two occasions recently, and an anticoccidial index where: Index = (pen clopidol had been used many years ago, but weight Day 6/pen weight Day 0) - (average decoquinate had not been employed. dropping pan score/10); as described by Jeffers (1974). The index range (UMU UMI) was assumed to represent the upper Parasites and lower limits of possible drug activity. Fecal droppings were collected from five The lower the index calculated by this farms at each complex and placed in 2.5% method the greater the pathological effects potassium dichromate. Oocysts present of the coccidia. within the samples were harvested and Isolates were classified as resistant, propagated in young chickens using proce- showing reduced sensitivity, or sensitive to dures that have been described elsewhere a particular drug if the anticoccidial index (Long et«/., 1976). No attempt was made to indicated a greater than 50% reduction in separate the species of Eimeria but, based efficacy, between 25 to 50% reduction in upon the location of lesions within the efficacy, or less than 25% reduction in intestines and the size of the oocysts, all 10 efficacy, respectively. The index for a 50% isolates consisted principally of both Ei- reduction in efficacy was calculated as meria acervulina and Eimeria maxima. (UMU - UMI)/2 + (UMI); whereas the index for a 25% reduction in efficacy was calculated as (UMU - UMI)/.75 + (UMI). Experimental Design Mean limiting indices for categories of Experiments included 1) medicated in- resistance for each complex are given in fected birds, 2) unmedicated infected birds Table 1. (UMI), and 3) unmedicated uninfected control birds (UMU). Two trials were carried out using five isolates from the Statistical Analysis respective complexes. For each isolate there In order to assess the overall efficacy of the drugs, observations for the 10 isolates from Complexes A and B were pooled for 2 statistical analysis (n = 10). Data were Rh6ne-Poulenc, Atlanta, GA 30350. 3 analyzed by one-way analysis of variance Hoffmann-La Roche Inc., Nutley, NJ 07110. 4 Elanco Products Co., Division of Eli Lilly and Co., (PROC ANOVA procedure of SAS® softIndianapolis, IN 46285. ware, SAS Institute, 1988) and means ^American Cyanamid Co., Agricultural Division, separated using the method of least square Wayne, NJ 07470. 6 differences (P < .05). Agri-Bio Corp., Gainesville, GA 30503.

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TABLE 1. Limiting indices used to define the drug sensitivity classification of Eitneria isolates Anticoccidial indices1 Source of isolates

UMI index2

UMU index3

Resistant4

Reduced sensitivity5

Sensitive6

Complex A Complex B

1.194 1.070

1.896 1.726

<1.546 <1.398

1.546 to 1.722 1.398 to 1.562

>1.722 >1.562

1

Average values for five farms at each complex. Unmedicated infected index. 3 Unmedicated uninfected index. 4 Greater than 50% reduction in the anticoccidial index. 'Between 25 and 50% reduction in the anticoccidial index. 6 Less than 25% reduction in the anticoccidial index. 2

There was no interaction between treatment and complex (P > .317); therefore, data for isolates from both complexes were combined. The mean anticoccidial indices for birds medicated with monensin, narasin, and salinomycin were not different from that of UMI birds (Figure 1). Birds given clopidol, decoquinate, robenidine, and lasalocid had significantly higher indices than the UMI birds (but lower indices than the UMU controls). The index for birds medicated with lasalocid was greater than that of birds given monensin, narasin, and salinomycin, but lower than that of birds given clopidol and robenidine. Classification of the sensitivity of isolates based upon the anticoccidial index is given in Table 2. All isolates were resistant to monensin, narasin, and salinomycin, but only six isolates were resistant to lasalocid. All isolates from Complex B were sensitive to robenidine, but three of the five isolates from Complex A were resistant to this drug. Most isolates were sensitive, or showed reduced sensitivity to clopidol and decoquinate. DISCUSSION

Monensin, salinomycin, and narasin were ineffective against the isolates obtained in this study. Monensin and salinomycin had been used extensively at both complexes, suggesting that resistance had been acquired as a result of exposure

to medication, but narasin had been used on only a single occasion. Cross resistance (presumed to be due to a common mode of action and mechanism of resistance) has been reported between isolates resistant to monensin, salinomycin, and narasin, thus explaining the lack of efficacy of the latter drug (Jeffers, 1984; Stallbaumer and Daisy, 1988). Lasalocid was more effective than the other ionophores, although six isolates were classified as resistant and four as showing reduced sensitivity to the drug. Lasalocid had not been used at either complex in this study. The situation regarding cross resistance between lasa-

2.00

CLO

DEC

LAS

MON

NAR

ROB

SAL

UMI

UMU

FIGURE 1. Effects of drugs on birds given isolates of Eimeria obtained from two broiler complexes. Each observation represents the mean anticoccidial index (ACI) + SEM for 10 isolates from Complexes A and B (5 from each complex) that were pooled for statistical analysis (n = 10). Means with no common letter are significantly different (P < .05). CLO = clopidol; DEC = decoquinate; LAS = lasalocid; MON = monensin; NAR = narasin; ROB = robenidine; SAL = salinomycin; UMI = unmedicated infected; UMU = unmedicated uninfected.

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RESULTS

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ANTICOCCIDIAL DRUG SENSITIVITY 1

TABLE 2. Sensitivity of isolates of Eimeria to anticoccidial drugs Complex B

Complex A Drug

A

B

C

D

E

F

G

H

J

K

Clopidol Decoquinate Lasalocid Monensin Narasin Robenidine Salinomycin

R R RS R R S R

S RS R R R R R

RS S RS R R R R

S S RS R R S R

RS RS R R R R R

RS RS R R R S R

S S R R R S R

RS S R R R S R

R RS RS R R S R

S RS R R R S R

locid and monensin, salinomycin, or nara- clopidol and decoquinate. Clopidol had sin is uncertain; some workers have found been employed on two occasions recently that resistance to these drugs is shared, at Complex A but had not been used for whereas others have reported that many years at Complex B. Decoquinate lasalocid is able to control strains resistant had not been employed for at least 10 yr at to other ionophores (reviewed by Jeffers, Complex A and had never been used at 1989). Isolates of Eimeria tenella from sites Complex B. Resistance to these two drugs where monensin but not lasalocid had was once widespread (Jeffers, 1974), but it been used were resistant to both drugs is not known whether resistant strains (Chapman and Shirley, 1989). The concen- were present at the farms investigated tration of lasalocid employed, however, here. Chapman (1989) found that most was 90 ppm, whereas in the present study isolates of E. tenella examined were sensi100 ppm was used. It is possible that tive to clopidol and all were sensitive to lasalocid is more effective at the higher methyl benzoquate (cross resistance occurs concentration. in isolates resistant to methyl benzoquate Robenidine had been used on two and decoquinate). It is possible that if occasions at Complex B, but all the isolates anticoccidial drugs are not used, restorafrom this complex were sensitive to the tion of sensitivity may eventually occur, drug. At Complex A, however, robenidine perhaps by the replacement of drughad not been used for at least 10 yr and resistant with drug-sensitive strains. yet three of the five isolates were classified Isolates from all farms at both comas resistant. No correlation was found, plexes were resistant to monensin and therefore, between the extent of drug use salinomycin. In the case of drugs that have and the occurrence of resistance. Acquisi- been used extensively, examination of tion of resistance was considered to be the isolates from one or two farms may, principal reason for the demise of robeni- therefore, give results applicable to the dine shortly after its introduction in the complex as a whole. Isolates varied in early 1970s, although this was not their sensitivity to robenidine, clopidol, documented at the time. It is possible that and decoquinate. In the case of drugs that at Complex B exposure to medication may have been used infrequently, examination have been of insufficient duration to of isolates from at least five farms would permit the selection of strains resistant to be necessary to establish the spectrum of robenidine. At Complex A, however, the drug sensitivity. results suggest that resistant strains may Sensitivity studies can indicate whether persist for a long time following cessation resistance is present to the drugs in of use of the drug. commercial use and whether other comThe majority of isolates were either pounds may be more effective. In practice, sensitive or showed reduced sensitivity to the number of isolates and farms that can

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1 Sensitivity of isolates to drugs was based upon calculation of an anticoccidial index. Isolates were classified as: R, resistant, greater than 50% reduction in efficacy; RS, showing reduced sensitivity, between 25 and 50% reduction in efficacy; and S, sensitive, less than 25% reduction in efficacy.

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Jeffers, T. K., 1984. Correlated responses of coccidia to polyether ionophorous antibiotics. Pages 589-591 in: Proceedings of the 17th World's Poultry Congress, August 8-12, 1984, World's Poultry Science Association, Helsinki, Finland. Jeffers, T. K., 1989. Anticoccidial drug resistance: a ACKNOWLEDGMENTS review with emphasis on the polyether ionophores. Pages 295-308 in: Coccidia and IntestiThis work was supported, in part, by a nal Coccidiomorphs. Proceedings of the Vth grant from Rh6ne-Poulenc, Inc., Atlanta, International Coccidiosis Conference, October GA 30350. The assistance of Z. B. Johnson 17-20,1989. P. Yvore, ed. Institut National de la is gratefully acknowledged. Recherche Agronomique, Paris, France. Long, P. L., L. P. Joyner, B. J. Millard, and C. C. Norton, 1976. A guide to laboratory techniques REFERENCES used in the study and diagnosis of avian coccidiosis. Folia Vet. Lat. 6:201-217. Chapman, H. D., 1989. Sensitivity of field isolates of Eimeria tenella to anticoccidial drugs in the National Research Council, 1984. Nutrient Requirements of Poultry. 8th rev. ed. National Academy chicken. Res. Vet. Sci. 47:125-128. Press, Washington, DC. Chapman, H. D., and M. W. Shirley, 1989. Sensitivity of field isolates of Eimeria species to monensin SAS Institute, 1988. SAS/STAT® User's Guide: Version 6. Fourth Edition. Vol. 1. SAS Institute Inc., and lasalocid in the chicken. Res. Vet. Sci. 46: Cary, NC. 114-117. Jeffers, T. K., 1974. Eimeria acervulina and E. maxima:Stallbaumer, M., and K. J. Daisy, 1988. The effects of monensin, narasin, salinomycin, and nicarbazin Incidence and anticoccidial drug resistance of on field strains of chicken coccidia. Avian isolants in major broiler-producing areas. Avian Dis. 18:332-342. Pathol. 17:793-301.

be investigated will be determined by the costs of the studies and the available facilities.

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