Studies on the sensitivity of field isolates of eimeria maxima to combinations of anticoccidial drugs

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1 Avian Pathology ISSN: (Print) (Online) Journal homepage: Studies on the sensitivity of field isolates of eimeria maxima to combinations of anticoccidial drugs H.D. Chapman To cite this article: H.D. Chapman (1980) Studies on the sensitivity of field isolates of eimeria maxima to combinations of anticoccidial drugs, Avian Pathology, 9:1, 67-76, DOI: / To link to this article: Published online: 12 Nov Submit your article to this journal Article views: 145 View related articles Citing articles: 12 View citing articles Full Terms & Conditions of access and use can be found at

2 Avian Pathology, 9: 67-76, 1980 STUDIES ON THE SENSITIVITY OF FIELD ISOLATES OF EIMERIA MAXIMA TO COMBINATIONS OF ANTICOCCIDIAL DRUGS H.D. CHAPMAN Houghton Poultry Research Station, Houghton Huntingdon, Cambs. PE17 2DA, England SUMMARY 1. The sensitivity of isolates of E. maxima to methyl benzoquate, clopidol, Lerbek, Amprolmix, Pancoxin and Supacox was studied. 2. Resistance to methyl benzoquate was widespread in isolates obtained from sites where broiler chickens were reared but isolates obtained from breeder farms were sensitive to the drug. Resistance to clopidol was found in all isolates examined. s resistant to both these drugs were also resistant to Lerbek but where sensitivity to one ingredient was present then Lerbek retained its efficacy. 3. All isolates were resistant to Amprolmix and Pancoxin although most were sensitive to Supacox. When given to chickens medicated with Amprolmix or Supacox isolates from breeder farms caused significantly lower weight gains than from broiler farms. 4. An attempt is made to correlate these observations with the history of medication at the sites. 5. No differences in pathogenicity were found between any of the isolates studied. INTRODUCTION Several advantages result from the use of combinations of different drugs to control coccidiosis. One advantage is that the spectrum of activity may be increased by combining two drugs which have preferential activity against different species. Thus, amprolium, which is not very active against the intestinal species oleimeria was recommended to be used with sulphaquinoxaline as mixtures of ppm amprolium and 80 ppm sulphaquinoxaline (Davies and Joyner, 1963) and Long (1963) found 60 ppm amprolium and 60 ppm sulphaquinoxaline effective. Currently in the United Kingdom, mixtures of amprolium (125 ppm) and ethopabate (8 ppm) or amprolium (100 ppm), sulphaquinoxaline (60 ppm) and ethopabate (8 ppm) are used. Received 18 July 1979 Accepted 14 September 1979

3 68 H.D. Chapman A second advantage is that mixtures of sulphonamides and pyrimidine compounds such as pyrimethamine were found to potentiate one another (Kendall and Joyner, 1956). The risk of toxicity due to the sulphonamide was therefore reduced since activity could be obtained at a lower dose than with either drug separately. Potentiation has been demonstrated between sulphaquinoxaline and ethopabate (McManus et al, 1967) and between quinolones and clopidol (Challey and Jeffers, 1973; Ryley, 1975). Recently a mixture of a quinolone (methyl benzoquate 8.35 ppm) and clopidol (100 ppm) has been introduced in the United Kingdom. A further advantage might be to reduce the problem of drug resistance since the chances of selecting mutants resistant to a combination of drugs used simultaneously is less than if those drugs are used alone (Bryson and Szybalski, 1955). For this reason mixtures of drugs have been used in the therapy of tuberculosis and malaria (Peters et al., 1973). Norton and Joyner (1975) obtained resistance to 10 ppm methyl benzoquate or 125 ppm clopidol after three passages of E. maxima but were later unsuccessful with a mixture of 8.35 ppm methyl benzoquate and 100 ppm clopidol (Joyner and Norton, 1978). They suggested that the difficulty in obtaining simultaneous resistance to these two drugs might offer a practical advantage. This advantage in the use of mixtures will depend upon whether resistance to the individual ingredients is already present since many compounds have been previously employed independently. In this study the sensitivity of field isolates of E. maxima to various combinations of anticoccidial drugs has been studied. s were obtained from environments where different compounds had been used thus permitting a correlation of drug resistance with the history of medication. MATERIALS AND METHODS Animals Details of animals used, maintenance and allocation to cages have been previously described (Chapman, 1979). Drugs Drugs were included in the diet at the concentrations (parts per million) indicated, 2 days before inoculation of birds with oocysts. Clopidol (100) and methyl benzoquate (8.35) were used at the concentrations recommended for use in Lerbek. Other drugs studied included: Lerbek 1, Amprolmix 2 (amprolium ethopabate 8), Pancoxin 2 (amprolium sulphaquinoxaline 60 + ethopabate 5) and Supacox 2 (amprolium sulphaquinoxaline 60 + ethopabate 5 + pyrimethamine 5). Infective material Details of the origin and methods of purification of the isolates used in this study have previously been described (Chapman, 1979). s are classified according to whether they were obtained from sites where broiler or breeder chickens were reared. Design of experiments Two experiments were carried out. In the first the response of isolates to clopidol, methyl benzoquate and Lerbek was examined. In the second experiment the response of the same isolates to Amprolmix, Pancoxin and Supacox was investigated. Each experiment was repeated three times and consisted of two parts in which different isolates were compared. In the first part, isolates from broiler farms (Ov, Ct, Sm, Wh), 1 Dow Chemical Co. Ltd. 2 Merck Sharp & Dohme Ltd.

4 Sensitivity of E. maxima to anticoccidial drugs 69 isolates from breeder farms (Cl, Eh, Dw) and the Houghton strain off. maxima were compared. In the second part broiler isolates (Fn, Bl, Ln, Br, Gr) and breeder isolates (Wt, Ch, Pr) were compared. Each part included two groups of five medicated and non-medicated inoculated birds and four groups of five non-medicated non-inoculated controls. Birds were inoculated with 10 5 oocysts of each isolate and weighed and killed 8 days later. Weight gains between 2 days before and 8 days after inoculation were calculated and results analysed by Student's T test of treatment means. History of medication At the time when the isolates were collected in 1977 monensin was in use at the broiler sites and dinitro-o-toluamide at the breeder sites. Mixtures containing amprolium had been extensively used in the breeder birds, mostly in the form of Pancoxin, containing amprolium, sulphaquinoxaline and ethopabate, which had been used for approximately 3 to 4 years until A mixture based on 6% amprolium and 0.38% ethopabate had occasionally been used in some units for treatment. For broiler chickens Pancoxin had also been used for 3 to 4 years and Supacox, containing amprolium, ethopabate, sulphaquinoxaline and pyrimethamine, had been in use at all broiler sites for 10 months. Clopidol had been in use at broiler sites only, for approximately 3 years. At some sites a quinolone had been used for a limited period of 6 months. Lerbek had not been used for either broilers or breeders. Definition of resistance The weight gain of medicated inoculated birds was compared with the non-medicated inoculated and uninoculated controls. Where weight gain was not significantly different from the non-medicated, non-inoculated birds (p > 0.05), a strain was judged sensitive (S). Where weight gain was not significantly different from non-medicated inoculated controls, a strain was considered resistant (R). Where weight gains were significantly different from both control groups (P <0.05), strains were judged partially resistant (PR). Pathogenicity The pathogenicity of the isolates was compared by examining the weight gains of nonmedicated chickens inoculated with 10 s oocysts; included in these results are data previously obtained (Chapman, 1979) for chickens of the same age and breed kept in the same conditions. RESULTS In broiler isolates resistance to clopidol, methyl benzoquate and Lerbek was widespread (Table 1 and 2). All nine isolates were either resistant or partially resistant, to clopidol. Six and seven isolates respectively were resistant or partially resistant to methyl benzoquate and Lerbek. Two of the broiler isolates sensitive to methyl benzoquate (Ln, Gr) were also sensitive to Lerbek. Five of six breeder isolates proved resistant or partially resistant to clopidol. All breeder isolates were sensitive to methyl benzoquate and Lerbek. The Houghton strain was sensitive to all three drugs (Table 1). Both broiler and breeder isolates were resistant to Amprolmix and Pancoxin (Tables 3 and 4). Nine broiler isolates were examined and seven proved to be sensitive to Supacox. Five of the six breeder isolates were also sensitive to this drug. The Houghton strain was sensitive to Supacox and Pancoxin but resistant to Amprolmix (Table 3). The weight gains of birds infected with broiler and breeder isolates were combined and the differences between their responses to different drugs compared by Student's't'

5 70 H.D. Chapman Table 1. Experiment 1: The effect of three drugs upon the weight gain of chickens inoculated with 10 s oocysts of eight isolates of E. maxima. Ov Sm Wh Cl Dw H Broiler Breeder Clopidol 69 R 54 R 83 R 106 Pr 91 R 85 R 113Pr 118 S Weight gain (g) a Methyl Lerbek benzoquate 80 R 56 R 74 R 124 S 116 S 124 S 121 S 136 S 84 R 72 R 71 R 115 Pr 140 S 132 S 148 S 149 S Weight gain between 2 days before and 8 days after infection. Weight gain of non-medicated, non-inoculated control = 146 ± 4.3 R = resistant; Pr = partially resistant; S = sensitive Standard error of each treatment mean = 8.6 No drug Table 2. Experiment 1: The effect of three drugs upon the weight gain of chickens inoculated with 10 s oocysts of eight isolates of E. maxima. Fn B1 Ln Br Gr Wt Ch Pr Broiler Breeder Clopidol 86 R 71 R 111 Pr 87 R 79 R 134 S 109 Pr 77 R Weight gain (g) a Methyl Lerbek benzoquate 75 R 64 R 131 S 99 Pr 125 S 122 S 146 S 153 S 88 R 70 R 136 S 96 R 142 S 141 S 141 S 147 S Weight gain between 2 days before and 8 days after infection. Weight gain of non-medicated, non-inoculated control = 149 ± 4.0. R = resistant; Pr = partially resistant; S = sensitive. Standard error of each treatment mean = 8.0. No drug test (Table 5). In comparison with breeder isolates infection with broiler isolates resulted in significantly lower weight gains with methyl benzoquate and Lerbek. The weight gain of birds given breeder isolates, however, was significantly lower than birds given broiler isolates with Amprolmix and Pancoxin. No significant difference was found with Supacox. The weight gain of chickens infected with the different isolates in the absence of drug is presented in Table 6. Differences between the eight isolates within each set were not significant.

6 Sensitivity of E. maxima to anticoccidial drugs 71 Table 3. Experiment 2: The effect of three drugs upon the weight gain of chickens inoculated with 10 s oocysts of eight isolates of E. maxima. Amprolmix Weight gain (g) a Pancoxin Supacox No drug Ov ^ Sm Wh Cl Dw H Broiler Breeder 85 R 93 R 89 R 91 R 60 R 60 R 97 R 90 R 104 R 95 R 104 R 111 R 77 R 80 R 89 R 159 S 110R 134 S 155 S 131 S 140 S 126 Pr 152 S 146 S Weight gain between 2 days before and 8 days after infection. Weight gain of non-medicated, non-inoculated control = 1S9 ± 4.7. R = resistant; Pr = partially resistant; S = sensitive. Standard error of each treatment mean = 9.3. Table 4. Experiment 2: The effect of three drugs upon the weight gain of chickens inoculated with 10 s oocysts of eight isolates of E. maxima. Fn Bl Ln Br Gr Wt Ch Pr Broiler Breeder Amprolmix 107 R 97 R 107 R 93 R 100 R 82 R 76 R 84 R Weight gain (g) a Pancoxin 106 R 107 R 120 R 107 R 98 R 69 R 87 R 73 R Supacox 136 S 120 Pr 156 S 139 S 149 S 143 S 140 S 150 S Weight gain between 2 days before and 8 days after infection. Weight gain of non-medicated, non-inoculated control = 162 ± 5.0. R = resistant; Pr = partially resistant; S = sensitive. Standard error of each treatment mean = 9.9. No drug

7 72 H.D. Chapman Table 5. Comparison of the weight gain of chickens medicated with different anticoccidial drugs and inoculated with 10 s oocysts of isolates of E. maxima obtained from either broiler or breeder sites. Drug Clopidol Methyl benzoquate Lerbek Amprolmix Pancoxin Supacox Broiler Weight gain (g) a Breeder Differences b Weight gain between 2 days before and 8 days after infection. Difference ± standard error ± ± ± ± ± 7.0 Table 6. Pathogenicity of six isolates ofe. maxima: comparisons of weight gain after inoculation with 10 s oocysts. Ov Ct Sm Wh Cl Eh Dw H Weight gain a Fn Bl Ln Br Gr Wt Ch Pr Significance NS 0.01<P< <f< <P<0.01 P< NS Weight gain b Each observation is the mean weight gain of 12 groups of birds measured between 2 days before and 8 days after infection, a Standard error of mean = 5.0. b Standard error of mean = 5.2. DISCUSSION Resistance to methyl benzoquate was widespread in broiler isolates but breeder isolates were sensitive to the drug. This may be correlated with the use of these compounds since a quinolone had been used for broiler but not for breeder chickens. Jeffers (1974) found that the incidence of resistance of field strains of E. maxima from broiler farms to the quinolone anticoccidials buquinolate and decoquinate was 62.5 and 73.7% respectively. Resistance to clopidol was also widespread. Both broiler and breeder isolates proved to be resistant despite the fact that the drug had never been used at breeder sites. The Houghton strain was sensitive although the weight gain was lower than in the non-infected control birds. This strain has been maintained in the laboratory and not exposed to anticoccidial drugs. This suggests that the resistance shown by these isolates may partly be due to lack of efficacy at the concentration of drug employed in the experiments. The recommended concentration of clopidol is 125 ppm when used alone whereas

8 100 ppm was used in this study. Sensitivity of maxima to anticoccidial drugs 73 Ryley and Betts (1973) found that clopidol at 125 ppm prevented weight loss and mortality in chickens but that oocyst production was not entirely suppressed; slightly depressed weight gain and increased oocyst production was noted at 62.5 ppm. Norton and Joyner (1975) infected birds fed clopidol at 62.5 ppm with 320,000 oocysts of the Weybridge strain of E. maxima and found that two passages were required before sufficient oocysts could be recovered to permit passage of the strain at a higher concentration. In an earlier study, however, weight loss and oocyst production was found with 125 ppm clopidol in birds inoculated with the same dose of oocysts (Norton and Joyner, 1968). In a further study 62 ppm was found ineffective against this strain (Joyner and Norton, 1978). Jeffers (1974) noted that 28.9% and 40.7% of field isolates off. maxima and E. acervulina respectively were resistant to clopidol. A significantly higher percentage of strains was resistant where clopidol was in use at the time samples were collected. In the experiments reported here, no correlation between drug usage and resistance was found. s resistant to both methyl benzoquate and clopidol were also resistant to Lerbek. Two broiler and six breeder isolates were controlled by methyl benzoquate and also proved sensitive to Lerbek suggesting that a drug mixture will retain efficacy where sensitivity to one ingredient is present. Joyner and Norton (1978) found that the control by Lerbek against a clopidol or methyl benzoquate resistant line of E. maxima was equivalent to the control by methyl benzoquate or clopidol respectively used alone against those lines. Lerbek was ineffective against a line made resistant to both drugs. Joyner and Norton (1978) have also shown that whilst selection of resistance to methyl benzoquate and clopidol used simultaneously is difficult, no difficulty is found in producing a doubly resistant strain to the recommended level of drug by initiating selection in a strain already resistant to one component. The high incidence of strains resistant to both methyl benzoquate and clopidol reported by Jeffers (1974) and found here suggests that in the field such selection has already taken place. Resistance to Amprolmix and Pancoxin was found in all broiler and breeder isolates. Failure of Amprolmix to control the Houghton strain of E. maxima which has never been exposed to the drug would support the contention of Jeffers (1974) that the poor response of field strains oie. maxima to drugs based upon a mixture of 100 ppm amprolium and 4 ppm ethopabate (Amprol Plus) is due to limited anticoccidial efficacy rather than drug resistance. Infection with breeder isolates resulted in significantly lower weight gains than broiler isolates with Amprolmix and Pancoxin. This may be due to the more recent use of Pancoxin in breeder birds. The poor response of Amprolmix may therefore be partly due to acquired drug resistance as well as limited efficacy. A survey carried out by Hodgson et al. (1969) over a 3 year period from revealed a significant increase in strains off?, maxima resistant to a mixture of 80 ppm amprolium, 60 ppm sulphaquinoxaline and 5 ppm ethopabate. The incidence of isolates resistant to the drug was 18.8, 41.9 and 71.9% in successive years. In this study none of the isolates was sensitive to Pancoxin, reflecting the wide use of this drug. Long et al. (1975) found that the incidence of oocysts in the litter from a broiler

9 74 H.D. Chapman farm where Supacox was in use was similar to that of broiler farms where clopidol was employed. Numbers of oocysts were reduced when monensin was substituted for Supacox. Three of the isolates used in the present study were obtained from broiler houses included in Long's survey and all proved to be resistant to clopidol. The isolates were sensitive to monensin although even with this drug oocyst production of isolates that had been exposed to monensin was higher than in unexposed isolates (Chapman, 1979). The presence of large numbers of oocysts in the litter from a broiler house may therefore indicate the presence of a resistant population of coccidia. The high number of oocysts reported by Long et al. (1975) from a farm where Supacox had been used could be due to the presence of coccidia resistant to this drug. Most isolates examined in this study were sensitive to Supacox suggesting that the drug has not been used long enough at the sites from which these isolates had been obtained for resistance to develop. The finding of one resistant and two partially resistant isolates suggests that resistance will ultimately develop to this drug also.norton and Hein (1976) reported that the Houghton strain of E. maxima was less pathogenic than the Weybridge and Norwich strains. Jeffers (1978), however, found no significant differences in the pathogenicities of six strains of this species and no significant difference in the pathogenicity of sixteen isolates was found here. A cknowledgements I would like to thank Mrs. M. Shaw for her assistance and Mr. J.G. Rowell, ARC. Statistics Group, for his collaboration and helpful advice. REFERENCES Bryson, V. and Szybalski, W. (1955). Microbial drug resistance. Advances in Genetics, 7:1-46. Challey, J.R. and Jeffers, T.K. (1973). Synergism between 4-hydroxy-quinoline and pyridone coccidiostats. Journal of Parasitology, 59: Chapman, H.D. (1979). Studies on the sensitivity of recent field isolates of E. maxima to monensin. Avian Pathology, 8: Davies, S.F.M. and Joyner, L.P. (1963). Design therapy for the control of species of Eimeria in the domestic fowl. Journal of Comparative Pathology, 73: Hodgson, J.N., Ball, S.J., Ryan, K.C. and Warren, E.W. (1969). The incidence of drug resistant strains of Eimeria in chickens in Great Britain, British Veterinary Journal, 125: Jeffers, T.K. (1974). Eimeria acervulina and E. maxima: Incidence and anticoccidial drug resistance of isolants in major broiler-producing areas. Avian Diseases, 18: Jeffers, T.K. (1978). Genetics of coccidia and the host response. In: 'Avian Coccidiosis'. 13th Poultry Science Symposium. Ed. Long, P.L., Boorman, K.N., and Freeman, B.M. British Poultry Science Ltd. Joyner, L.P. and Norton, C.C. (1978). The activity of methyl benzoquate and clopidol against Eimeria maxima: synergy and drug resistance. Parasitology, 76: Kendall, S.B. and Joyner, L.P. (1956). The synergism between pyrimethamine and sulphadimethyl pyrimidine in the control of Eimeria tenella. Journal of Comparative Pathology and Therapeutics, 66: Long, P.L. (1963). The effect of a combination of sulphaquinoxaline and amprolium against different species of Eimeria in chickens. Veterinary Record, 75: Long, P.L., Tompkins, R. V. and Millard, B.J. (1975). Coccidiosis in broilers: evaluation of infection by the examination of broiler house litter for oocysts. Avian Pathology, 4: McManus, E.C., Oberdick, M.T. and Cuckler, A.C. (1967). Response of six strains of Eimeria brunetti to two antagonists of para aminobenzoic acid. Journal of Protozoology, 14: Norton, C.C. and Hein, H.E. (1976). Eimeria maxima: a comparison of two laboratory strains with a fresh isolate. Parasitology, 72:

10 Sensitivity of E. maxima to anticoccidial drugs 75 Norton, C.C. and Joyner, L.P. (1968). Coccidiostatic activity of meticlorpindol. Effectiveness against single infections of five species of Eimeria in the domestic fowl. Veterinary Record, 83: Norton, C.C. and Joyner, L.P. (1975). The development of drug-resistant strains of Eimeria maxima in the laboratory. Parasitology, 71: Peters, W., Portus, J.H. and Robinson, B.L. (1973). The chemotherapy of rodent malaria, XVII. Dynamics of drug resistance, Part 3: influence of drug combinations on the development of resistance to chloroquine in P. berghei. Annals of Tropical Medicine and Parasitology, 67: Ryley, J.F. (1975). Lerbek, a synergistic mixture of methyl benzoquate and clopidol. Parasitology, 70: Ryley, J.F. and Betts, M.J. (1973). Chemotherapy of chicken coccidiosis-advances in Pharmacology and Chemotherapy, 11: RESUME Etude de la sensibilité de souches d'eimeria maxima vis-à-vis d'associations de produits anticoccidiens 1. La sensibilité de souches d'eimeria maxima a été étudiée avec les produits suivants: Méthyl benzoquate, Clopidol, Lerbek, Amprolmix, Pancoxin et Supacox. 2. La résistance au Méthyl benzoquate a été très fréquente en ce qui concerne les souches provenant de fermes où des poulets de chair avaient été élevés, alors que celles provenant d'élevages de reproduction étaient sensibles au produit. La résistance au Clopidol fut trouvée pour tous les isolements réalisés. Les souches résistantes à ces 2 produits, l'étaient aussi pour le Lerbek, mais lorsqu'il y avait sensibilité vis-à-vis d'un des composants, le Lerbek conservait son efficacité. 3. Toutes les souches isolées se sont révélées résistantes à l'amprolmix et au Pancoxin, bien que la plupart étaient sensibles au Supacox. Lorsque les souches étaient administrées à des poulets traités avec l'amprolmix et le Supacox, celles provenant des fermes de reproducteurs entraînaient une diminution de la croissance pondérale signifivativement plus faible que celles provenant des fermes de poulets de chair. 4. Il a été tenté d'établir une corrélation entre ces observations et l'historique des médications dans les différentes fermes. 5. Aucune différence de pouvoir pathogène n'a été trouvée entre les différentes souches isolées. ZUSAMMENFASSUNG Untersuchungen über die Empfindlichkeit von Feldisolaten von Eimeria maxima gegen Anticoccidiostatika-Kombinationen 1. Die Empfindlichkeit der von E. maxima gegen Methyl benzoquate, Clopidol, Lerbek, Amprolmix, Pancoxin und Supacox wurde untersucht. 2. Eine Resistenz gegen Methyl benzoquate war bei n aus Broileraufzuchtbeständen weit verbreitet, während aus Zuchtgeflügelherden gegen die Droge empfindlich waren. Eine Resistenz gegen Clopidol wurde bei allen untersuchten n festgestellt., die gegen die beiden Drogen resistent waren, waren es auch gegen Lerbek. Solange aber noch eine Empfindlichkeit gegen einen Bestandteil vorlage, behielt Lerbek seine Wirksamkeit. 3. Alle waren resistent gegen Amprolmix und Pancoxin obwohl die meisten

11 76 H.D. Chapman gegen Supacox empfindlich waren. Wenn Küken, die mit Amprolmix oder Supacox behandelt wurden, aus Zuchtbeständen verabreicht wurden, dann bewirkten diese eine signifikant niedrigere Gewichtszunahme als aus Broilerfarmen. 4. Es wird der Versuch unternommen, diese Beobachtungen mit der Geschichte der Arzneimittelversorgung dieser Herden in Einklang zu bringen. 5. Es wurden keine Unterschiede der Pathogenität zwischen den verschiedenen n beobachtet.