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Identification of Cross-Resistance and Multiple Resistance in Eimeria tenella Field Isolates to Commonly Used Anticoccidials in Pakistan
©2008 Poultry Science Association, Inc.
Identification of Cross-Resistance and Multiple Resistance in Eimeria tenella Field Isolates to Commonly Used Anticoccidials in Pakistan R. Z. Abbas,*1 Z. Iqbal,* Z.-D. Sindhu,* M. N. Khan,* and M. Arshad† *Department of Parasitology, and †Department of Microbiology, University of Agriculture, Faisalabad-38040-Pakistan Primary Audience: Flock Supervisors, Researchers, Feed Manufacturers
SUMMARY A battery trial was conducted to evaluate the drug sensitivity in Eimeria tenella field isolates against the commonly used anticoccidials salinomycin (60 ppm), maduramicin (5 ppm), and clopidol (125 ppm) in broiler chicks. These anticoccidials were mixed in feed at d 12 of age, and inoculation was given on d 14 of age. Drug sensitivity was determined by using the global index, which is composed of percentage weight gain, FCR, lesion score, oocyst index, and mortality percentage. In the present study, all the E. tenella isolates showed partial resistance against salinomycin, whereas varying degrees of sensitivity were observed against maduramicin and clopidol. Key words: Eimeria tenella, ionophore, anticoccidial, drug resistance, global index 2008 J. Appl. Poult. Res. 17:361–368 doi:10.3382/japr.2008-00027
DESCRIPTION OF PROBLEM Among various parasitic infections, coccidiosis is a major constraint for commercial poultry production. Coccidiosis is an intestinal infection that is caused by various species of intracellular protozoan parasites belonging to the genus Eimeria. In general, coccidiosis results in intestinal lesions, enteritis, and diarrhea [1], and extensive damage to the digestive tract may lead to death [2]. There are different species of avian coccidia, such as Eimeria tenella, Eimeria necatrix, Eimeria brunetti, Eimeria praecox, Eimeria acervulina, Eimeria mitis, and Eimeria maxima [3]. Each Eimeria species has a particular predilection site in the chicken digestive tract; for example, E. tenella attacks the cecum. However, the most common and pathogenic species 1
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that affects the poultry industry in Pakistan is E. tenella [4], which results in high mortality. Since the 1940s, anticoccidials have been introduced in increasing numbers; however, the emergence of drug resistance in coccidia, which, in due course, limits their use, is a great problem with most of the drugs [5, 6]. A quote from Schnitzer and Grunberg [7] aptly characterizes this problem: “Drug resistance has followed the development of chemotherapy like a faithful shadow.” It is becoming increasingly difficult to develop anticoccidial active ingredients that can replace old products. Coccidiosis appears to be no exception, and there is growing evidence that changes are occurring in the expected response to known coccidiostatic drugs. This phenomenon is now the subject of consideration.
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362 Information is not available in Pakistan regarding the development of resistance in Eimeria species against commonly used anticoccidial compounds. The reports available on the development of resistance in one country cannot be used as such to minimize the development of resistance and to plan for effective coccidiosis control strategies in other countries because of strain variations of coccidian species in different geographical locations and different schedules of using anticoccidials. Therefore, the present study has been undertaken to investigate resistance to the commonly used anticoccidials in the field isolates of E. tenella, because timely detection of resistance could direct the more rational use of drugs, help to improve the management of resistant parasites, and prolong the life of many chemicals.
MATERIALS AND METHODS Birds A total of 286 one-day-old Hubbard broiler chicks were purchased from a local hatchery [8]. Chicks were reared under standard management practices. All the chicks were kept on broiler starter ration to 2 wk of age and then fed a broiler finisher diet. Feed and water were provided ad libitum. Temperature was maintained at 85 to 90°F during the first week of age and was reduced by 5°F on a weekly basis. Lighting was provided for 24 h throughout the experimental period. All the birds were vaccinated for Newcastle disease on d 5, for infectious bursal disease on d 14, and for hydropericardium syndrome on d 18 of age. Parasite Three isolates of E. tenella were collected from poultry farms that were located distantly apart in Faisalabad district of Punjab, Pakistan (with a history of prophylactic anticoccidial medication failure). Coccidial oocysts were obtained from the ceca of infected chicks and were propagated in broiler chicks by giving an oral infection. After obtaining a sufficient amount of oocysts, they were sporulated by placing them in 2.5% K2Cr2O7 in the presence of suitable humidity and temperature. Sporulated oocysts were cleaned and counted by the McMaster
technique [9]. The required concentration of the sporulated oocysts (75,000/mL) was maintained with PBS. The sporulated oocysts were inoculated in chicks orally at 14 d of age. Drugs Salinomycin (60 ppm), maduramicin (5 ppm), and clopidol (125 ppm) were purchased from the market [10] and included in the feed at 12 d of age (2 d before inoculation of the birds with E. tenella sporulated oocysts), and medication was continued up to 7 d postinoculation of sporulated oocysts. Experimental Design A total of 286 chicks were divided into 13 groups of 22 chicks each at the age of 12 d. The birds were reared in battery pens on wire floors. All the birds were tagged to maintain their identity. The chicks in all the groups except group 13 were infected with 75,000 sporulated oocysts of E. tenella. The chicks in groups 1 to 4 were infected with isolate 1, those in groups 5 to 8 with isolate 2, and those in groups 9 to 12 with isolate 3. Groups 1, 5, and 9 were medicated with salinomycin; groups 2, 6, and 10 with maduramicin; and groups 3, 7, and 11 with clopidol. Groups 4, 8, and 12 served as infected, nonmedicated control groups for isolate 1, isolate 2, and isolate 3, respectively. Group 13 was kept as a noninfected, nonmedicated control (NNC). Evaluation of Sensitivity or Resistance Seventeen chicks from each group were weighed individually on the day of inoculation (14 d of age) and then reweighed on d 7 postinoculation (21 d of age). Percentage BW gain between these days was recorded. Five chicks from each group were sacrificed for postmortem examination at 7 d postinoculation (21 d of age). Cecal lesions were scored by the lesion scoring technique described by Johnson and Reid [11]. An oocyst index (0 to 5) was determined by microscopic examination of scrapings from the ceca of chicks sacrificed for lesion scoring at d 7 postinoculation [12]. Mortality was recorded throughout the experimental period, and the exact cause of mortality was confirmed by postmortem examination.
Abbas et al.: EIMERIA TENELLA RESISTANCE Stephen et al. [13] developed the global index (GI), calculated on the basis of weight gain (%), feed conversion (g/g), lesion scores, oocyst index, and mortality (%), by using the formula GI = %WGNNC − [(FM − FNNC) × 10] − (OIM − OIINC) − [(LSM − LSINC) × 2] − (%mortality/2), where GI is the global index, WG is weight gain, F is the FCR, OI is the oocyst index, LS is the lesion score, M is the medicated group, NNC is the noninfected, nonmedicated control group, and INC is the infected, nonmedicated control group. The drug resistance of each isolate of E. tenella against the respective anticoccidial drug was also determined by calculating the GI. In addition, the GI for each test group was calculated as a percentage of the GI for the NNC. The following 5 categories were used for testing resistance to anticoccidials: 1) very good efficacy, ≥90% GINNC; 2) good efficacy, 80 to 89% GINNC; 3) limited efficacy, 70 to 79% GINNC; 4) partially resistant, 50 to 69% GINNC; and 5) resistant, <50% GINNC. Statistical Analysis Data obtained on various parameters were analyzed by ANOVA, and the mean values were compared by Tukey’s test. The experimental unit was the bird. The means for each isolate were compared separately. The results were recorded as mean ± SEM, and the differences among group means were considered significant at P < 0.05.
RESULTS AND DISCUSSION Comparative results of the different parameters used to calculate the GI of salinomycin, maduramicin, and clopidol are summarized in Table 1. It is evident from this table that the average weight gain of the salinomycin-medicated groups did not differ significantly (P > 0.05) from the infected, nonmedicated control groups (INC), except in salinomycin-medicated isolate 1 (SAL 1). The average weight gain of the maduramicin-medicated groups was significantly (P < 0.05) greater than that of the infected, nonmedicated control groups (INC), except in maduramicin-medicated isolate 3 (MAD 3). However, the average weight gain of the clopidol-medicated groups was significantly
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(P < 0.05) greater than that of the respective infected, nonmedicated control groups (INC) for all the isolates. Although the clopidol-medicated groups tended to have greater weight gain in all 3 isolates compared with salinomycin, it was only significant in isolate 3 (CLOP 3), in which the maximum weight gain was found. The results of FCR (Table 1) revealed a pattern relatively similar to that of weight gain among the different isolates of E. tenella. Although a statistical comparison could not be made because of group feeding, the FCR values of medicated groups were numerically lower compared with the infected, nonmedicated groups. The lesion scores of the maduramicin- and clopidol-medicated groups for isolates 1 and 2 differed significantly (P < 0.05) from the lesion scores of the respective infected, nonmedicated groups, but all other medicated groups showed lesion scores similar (P > 0.05) to the respective infected, nonmedicated groups. The results of oocyst scores (Table 1) indicated a significant anticoccidial effect of the salinomycin, maduramicin, and clopidol in isolates 1 and 3 of E. tenella. The oocyst scores calculated for these 2 isolates were lower (P < 0.05) compared with their respective infected, nonmedicated groups. However, isolate 2 showed no difference (P > 0.05) in oocyst scores between the medicated and infected, nonmedicated groups for all the anticoccidials. Mortality (Table 1) in all the medicated groups was greatest in the salinomycin-medicated groups, followed by the maduramicin-medicated groups and the clopidol-medicated groups. However, the mortality percentage was greatest in the infected, nonmedicated groups compared with the medicated groups. Data on the considered criterion (i.e., GI) for the medicated groups (Table 2) compared with the negative and positive controls revealed that the 3 isolates of E. tenella included in the present study had the same level of susceptibility to salinomycin. All the isolates showed partial resistance against salinomycin. However, the isolates of E. tenella showed varying levels of susceptibility to maduramicin and clopidol. Isolate 2 was the most susceptible, followed by isolates 3 and 1, respectively, for maduramicin. Clopidol showed good efficacy against isolates 2 and 3, but isolate 1 showed partial resistance. It was
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Table 1. Comparative values of the mean (and SEM) weight gain, FCR, lesion score, oocyst index, and mortality percentage1
Group
2
SAL 1 MAD 1 CLOP 1 INC 1 SAL 2 MAD 2 CLOP 2 INC 2 SAL 3 MAD 3 CLOP 3 INC 3 NNC SEM
Weight gain (g) 199d 208cd 217cd 154e 212cd 253bc 248bc 174de 221cd 228bcd 264ab 195d 290a 13.83
Weight gain of NNC (%)
FCR3 (g/g)
Lesion score
Oocyst score
Mortality3 (%)
70 71 77 52 75 89 87 62 77 80 93 70 100 —
1.29 1.27 1.27 1.42 1.26 1.20 1.21 1.38 1.23 1.25 1.17 1.32 1.14 —
2.8abc 1.8bc 1.8bc 3.4a 2.4abc 1.6c 1.4c 3.2ab 2.6abc 1.6c 1.6c 3.0abc — 0.43
2.6bc 1.6cd 1.8bcd 4.6a 2.0bcd 2.2bcd 2.2bcd 3.0b 2.2bcd 1.4cd 2.6bc 4.2a — 0.40
13.63 9.09 9.09 22.72 9.09 4.54 4.54 18.18 9.09 9.09 4.54 13.63 0 —
a–e
Means sharing similar superscripts within a column do not differ (P < 0.05). Bird served as the experimental unit. 2 SAL 1 = salinomycin-medicated isolate 1; SAL 2 = salinomycin-medicated isolate 2; SAL 3 = salinomycin-medicated isolate 3; MAD 1 = maduramicin-medicated isolate 1; MAD 2 = maduramicin-medicated isolate 2; MAD 3 = maduramicin-medicated isolate 3; CLOP 1 = clopidol-medicated isolate 1; CLOP 2 = clopidol-medicated isolate 2; CLOP 3 = clopidol-medicated isolate 3; INC 1, 2, and 3 = infected, nonmedicated controls; NNC = noninfected, nonmedicated control. 3 Statistical analysis was not possible because of group feeding of chicks. 1
evident from the results (Table 2) that none of the E. tenella field isolates showed complete sensitivity or complete resistance to the 3 anticoccidials. However, isolate 1 showed cross-resistance between salinomycin and maduramicin (polyether ionophore antibiotics) and multiple resistance among salinomycin, maduramicin, and clopidol. Numerous anticoccidial drugs have been introduced since the end of the 1940s, and over time, resistance to all these anticoccidial drugs has developed in Eimeria species [14, 15]. Several reports are available regarding the development of resistance or loss of sensitivity to the anticoccidial drugs used in the present studies, that is, resistance to salinomycin [13, 16–20], maduramicin [13, 20–22], and clopidol [23]. Therefore, resistance against commonly used anticoccidials is a global issue. Although there are also an increasing number of reports on resistant Eimeria field isolates in countries near to Pakistan, such as India [19, 23–25] and China [17, 20, 26], this is the first study reported in Pakistan. Different formulae have been used by different researchers to find out the sensitivity or resistance to anticoccidial drugs. The older formu-
lae or indices used for determining sensitivity or resistance were the performance index [27] and the anticoccidial index [28–30]. In them, no importance was given to FCR, which is considered to be an important parameter in determining resistance or sensitivity to any anticoccidial drug, because feed costs constitute some 70% of the cost of producing broiler chickens [31]. Presently, the newly devised method of Stephen et al. [13] was used for calculating the GI to detect resistance to anticoccidials in Eimeria spp. In this formula, all 5 parameters—weight gain, FCR, lesion scores, oocyst scores, and mortality—have been given their due importance. The results obtained with this formula corresponded very well to clinical symptoms and postmortem findings. Therefore, the GI can be considered a suitable tool to describe anticoccidial resistance as the above-mentioned conclusive overall picture. Many researchers [13, 19, 32] have correlated the resistance results with practical field conditions with great success by using the GI. Similar to using different parameters to assess the resistance or sensitivity to anticoccidial drugs, using more than 1 field isolate of the Eimeria species is also necessary, because only 1 isolate of Eimeria species is insufficient
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Table 2. Global index and efficacy status of salinomycin, maduramicin, and clopidol against Eimeria tenella field isolates Group1
Global index2
Global index of NNC (%)
Efficacy status3
SAL 1 MAD 1 CLOP 1 SAL 2 MAD 2 CLOP 2 SAL 3 MAD 3 CLOP 3 INC 1 INC 2 INC 3 NNC
64.79 71.36 77.06 71.76 90.13 88.43 74.26 79.96 94.73 37.84 50.41 61.39 110.3
58.73 64.69 69.86 65.05 81.71 80.17 67.32 72.49 85.88 34.30 45.70 55.65 100
4 4 4 4 2 2 4 3 2 — — — —
1
SAL 1 = salinomycin-medicated isolate 1; SAL 2 = salinomycin-medicated isolate 2; SAL 3 = salinomycin-medicated isolate 3; MAD 1 = maduramicin-medicated isolate 1; MAD 2 = maduramicin-medicated isolate 2; MAD 3 = maduramicin-medicated isolate 3; CLOP 1 = clopidol-medicated isolate 1; CLOP 2 = clopidol-medicated isolate 2; CLOP 3 = clopidol-medicated isolate 3; INC 1, 2, and 3 = infected nonmedicated controls; NNC = noninfected, nonmedicated control. 2 Global index (GI) = %WGNNC − [(FM − FNNC) × 10] − (OIM − OIINC) − [(LSM − LSINC) × 2] − (%mortality/2), where WG is weight gain, F is the FCR, OI is the oocyst index, LS is the lesion score, M is the medicated group, NNC is the noninfected, nonmedicated control group, and INC is the infected nonmedicated control group. 3 Efficacy status was calculated as percentage of the GI for the NNC. The following 5 categories were used for testing resistance to anticoccidials: 1) very good efficacy, ≥90% GINNC; 2) good efficacy, 80 to 89% GINNC; 3) limited efficacy, 70 to 79% GINNC; 4) partially resistant, 50 to 69% GINNC; and 5) resistant, <50% GINNC.
to give a conclusive summary of the resistance under field conditions. The Eimeria field isolates of one species consist of several strains. Environmental selection pressures in different geographical locations, as well as the histories of drug use, may differ; therefore, strains resistant in one area may be sensitive in other areas. These strain variants may express different epitopes, leading to immunological diversity, as evidenced by a lack of cross-protection [33]. The resistance test thus gives an overview of the combined effects of all these strains. Sensitive, partially resistant, and resistant strains contribute to this conclusive picture. Therefore, it is not necessary for every resistant strain to have all resistances detected in the isolate [13]. In the present study, a relatively similar pattern was observed, in that all 3 field isolates of E. tenella showed partial resistance against only salinomycin, but in all other treated groups, all the isolates responded differently in terms of sensitivity against maduramicin and clopidol. None of the Eimeria isolates was found to have complete resistance, and none was fully sensitive to the 3 anticoccidials.
Results of the present study revealed partial resistance in all 3 isolates of E. tenella against salinomycin, but all the isolates showed varying degrees of sensitivity to maduramicin and clopidol, except isolate 1, which showed partial resistance against both maduramicin and clopidol. Previous works have also demonstrated the sensitivity of Eimeria isolates to maduramicin [34, 35]. The presence of resistance to salinomycin in all isolates may be due to the extensive use of this ionophore by the poultry feed mills, because this is the cheapest anticoccidial available in Pakistan [36]. Numerous reports [37–40] have also suggested that some Eimeria field isolates may show reduced sensitivity to the polyether ionophore anticoccidials, which have been used extensively since their discovery in 1971. In Pakistan, prophylaxis for coccidiosis is chiefly dependent on the selection of anticoccidial by the feed mills, and single-drug usage programs practice the use of anticoccidials. However, some feed manufacturing companies do not declare the type and quantity of anticoccidial used. Poultry farmers treat coccidiosis by using anticoccidial drugs in the drink-
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366 ing water and also use some anticoccidial feed premix. The medication may continue for 3 to 4 wk. In this way, there is prolonged medication with underdosing of anticoccidial drugs. Furthermore, poultry farmers do not have mixers for the proper mixing of anticoccidials in the feed. Therefore, the other possible reason may be the use of lower dose levels in poultry feed by feed manufacturers and improper mixing by the farmers. The reason for the absence of resistance to the ionophore maduramicin may be that these compounds have not been in use for long periods in Pakistan. Another reason might be the one suggested by Ruff and Danforth [41], that the particular mode of action of ionophores does not allow an easy emergence of resistance against this class of anticoccidials, and only a limited number of cases of complete resistance have been cited in the scientific literature. The partial resistance, or a tendency for the development of resistance, against ionophores in the current study may therefore be attributed to their use for more than a decade, which might have resulted in a gradual reduction in the sensitivity of coccidia. In addition, however, the possibility of cross-resistance among ionophores cannot be ruled out in view of their similar mode of action through the general mechanisms of altering ion transport and disrupting osmotic balance. The present results on ionophores substantiate the findings of Raether and Paeffgen [42], who acknowledged a high degree of cross-resistance between salinomycin and maduramicin. Yet Bedrnik et al. [43] found that maduramicin was effective in controlling isolates of Eimeria that were resistant to other ionophores. Therefore, the difference in the activity of the 2 ionophores (salinomycin and maduramicin) observed in this study may be due to the development of incomplete cross-resistance. For these reasons, forming generalizations about cross-resistance in the polyether ionophores is difficult and imprecise. Isolate 1 was also found to be partially resistant to the chemical anticoccidial clopidol. This loss of sensitivity of isolate 1 to both the ionophores and the chemical anticoccidial clopidol may be due to the development of multiple resistance, because genetic recombination is probably one mechanism by which resistance to
several drugs with different modes of action can arise in the field, as also reviewed by different researchers [15]. Because several reports have shown the presence of resistance in coccidia against ionophores, it might be questioned why these ionophores are still widely used worldwide. First, these anticoccidials have beneficial effects, such as the control of certain gram-positive bacteria. Second, these anticoccidials do not completely suppress the development of coccidia; resistance is usually partial and therefore results in the acquisition of natural immunity by the infected birds [43]. The development of partial resistance to ionophores and clopidol in E. tenella field isolates is also evident from our findings. However, detection of drug resistance in experimental trials does not mean that resistance occurs under field conditions; it only suggests a need to change the routinely used coccidiosis control programs to prolong the life span of anticoccidials, as also suggested by Peeters et al. [44]. The rotation (the use of different anticoccidial drugs among flocks) and shuttle programs (the common use of 2 or more drugs within a single flock) are routinely used to manage the resistance problem. The rotation of anticoccidials with the vaccine containing the anticoccidial drug-sensitive strain has been reported to enhance the sensitivity of Eimeria isolates from 25 to 100% [45]. In a similar way, diclazuril in shuttle programs with other chemical anticoccidials and ionophores is highly efficacious against different Eimeria spp. [46].
CONCLUSIONS AND APPLICATIONS
1. The straight use (single-drug use) of the studied anticoccidials—particularly salinomycin, but also the other 2 drugs, maduramicin and clopidol—should be minimized. 2. These anticoccidials should be used by rotating them with different anticoccidial drugs among flocks. 3. The other possible way to delay the development of resistance may be through the use of shuttle programs.
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JAPR: Research Report Proc. Vth Int. Coccidiosis Conf., Tours, France. INRA Publications, Versailles, France. 43. Chapman, H. D. 1999. Anticoccidial drugs and their effects upon the development of immunity to Eimeria infections in poultry. Avian Pathol. 28:521–535. 44. Peeters, J. E., J. Derijcke, M. Verlinden, and R. Wyffels. 1994. Sensitivity of avian Eimeria spp. to seven chemical and five ionophores anticoccidials in five Belgian integrated broiler operations. Avian Dis. 38:483–493. 45. Mathis, G. F., and C. Broussard. 2006. Increased level of Eimeria sensitivity to diclazuril after using a live coccidial vaccine. Avian Dis. 50:321–324. 46. Conway, D. P., G. F. Mathis, J. Johonson, M. Schwartz, and C. Baldwin. 2001. Efficacy of diclazuril in comparison with chemical and ionophorous anticoccidials against Eimeria spp. in broiler chickens in floor pens. Poult. Sci. 80:426–430.
Identification of Cross-Resistance and Multiple Resistance in Eimeria tenella Field Isolates to Commonly Used Anticoccidials in Pakistan R. Z. Abbas,*1 Z. Iqbal,* Z.-D. Sindhu,* M. N. Khan,* and M. Arshad† *Department of Parasitology, and †Department of Microbiology, University of Agriculture, Faisalabad-38040-Pakistan Primary Audience: Flock Supervisors, Researchers, Feed Manufacturers
SUMMARY A battery trial was conducted to evaluate the drug sensitivity in Eimeria tenella field isolates against the commonly used anticoccidials salinomycin (60 ppm), maduramicin (5 ppm), and clopidol (125 ppm) in broiler chicks. These anticoccidials were mixed in feed at d 12 of age, and inoculation was given on d 14 of age. Drug sensitivity was determined by using the global index, which is composed of percentage weight gain, FCR, lesion score, oocyst index, and mortality percentage. In the present study, all the E. tenella isolates showed partial resistance against salinomycin, whereas varying degrees of sensitivity were observed against maduramicin and clopidol. Key words: Eimeria tenella, ionophore, anticoccidial, drug resistance, global index 2008 J. Appl. Poult. Res. 17:361–368 doi:10.3382/japr.2008-00027
DESCRIPTION OF PROBLEM Among various parasitic infections, coccidiosis is a major constraint for commercial poultry production. Coccidiosis is an intestinal infection that is caused by various species of intracellular protozoan parasites belonging to the genus Eimeria. In general, coccidiosis results in intestinal lesions, enteritis, and diarrhea [1], and extensive damage to the digestive tract may lead to death [2]. There are different species of avian coccidia, such as Eimeria tenella, Eimeria necatrix, Eimeria brunetti, Eimeria praecox, Eimeria acervulina, Eimeria mitis, and Eimeria maxima [3]. Each Eimeria species has a particular predilection site in the chicken digestive tract; for example, E. tenella attacks the cecum. However, the most common and pathogenic species 1
Corresponding author: [email protected]
that affects the poultry industry in Pakistan is E. tenella [4], which results in high mortality. Since the 1940s, anticoccidials have been introduced in increasing numbers; however, the emergence of drug resistance in coccidia, which, in due course, limits their use, is a great problem with most of the drugs [5, 6]. A quote from Schnitzer and Grunberg [7] aptly characterizes this problem: “Drug resistance has followed the development of chemotherapy like a faithful shadow.” It is becoming increasingly difficult to develop anticoccidial active ingredients that can replace old products. Coccidiosis appears to be no exception, and there is growing evidence that changes are occurring in the expected response to known coccidiostatic drugs. This phenomenon is now the subject of consideration.
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362 Information is not available in Pakistan regarding the development of resistance in Eimeria species against commonly used anticoccidial compounds. The reports available on the development of resistance in one country cannot be used as such to minimize the development of resistance and to plan for effective coccidiosis control strategies in other countries because of strain variations of coccidian species in different geographical locations and different schedules of using anticoccidials. Therefore, the present study has been undertaken to investigate resistance to the commonly used anticoccidials in the field isolates of E. tenella, because timely detection of resistance could direct the more rational use of drugs, help to improve the management of resistant parasites, and prolong the life of many chemicals.
MATERIALS AND METHODS Birds A total of 286 one-day-old Hubbard broiler chicks were purchased from a local hatchery [8]. Chicks were reared under standard management practices. All the chicks were kept on broiler starter ration to 2 wk of age and then fed a broiler finisher diet. Feed and water were provided ad libitum. Temperature was maintained at 85 to 90°F during the first week of age and was reduced by 5°F on a weekly basis. Lighting was provided for 24 h throughout the experimental period. All the birds were vaccinated for Newcastle disease on d 5, for infectious bursal disease on d 14, and for hydropericardium syndrome on d 18 of age. Parasite Three isolates of E. tenella were collected from poultry farms that were located distantly apart in Faisalabad district of Punjab, Pakistan (with a history of prophylactic anticoccidial medication failure). Coccidial oocysts were obtained from the ceca of infected chicks and were propagated in broiler chicks by giving an oral infection. After obtaining a sufficient amount of oocysts, they were sporulated by placing them in 2.5% K2Cr2O7 in the presence of suitable humidity and temperature. Sporulated oocysts were cleaned and counted by the McMaster
technique [9]. The required concentration of the sporulated oocysts (75,000/mL) was maintained with PBS. The sporulated oocysts were inoculated in chicks orally at 14 d of age. Drugs Salinomycin (60 ppm), maduramicin (5 ppm), and clopidol (125 ppm) were purchased from the market [10] and included in the feed at 12 d of age (2 d before inoculation of the birds with E. tenella sporulated oocysts), and medication was continued up to 7 d postinoculation of sporulated oocysts. Experimental Design A total of 286 chicks were divided into 13 groups of 22 chicks each at the age of 12 d. The birds were reared in battery pens on wire floors. All the birds were tagged to maintain their identity. The chicks in all the groups except group 13 were infected with 75,000 sporulated oocysts of E. tenella. The chicks in groups 1 to 4 were infected with isolate 1, those in groups 5 to 8 with isolate 2, and those in groups 9 to 12 with isolate 3. Groups 1, 5, and 9 were medicated with salinomycin; groups 2, 6, and 10 with maduramicin; and groups 3, 7, and 11 with clopidol. Groups 4, 8, and 12 served as infected, nonmedicated control groups for isolate 1, isolate 2, and isolate 3, respectively. Group 13 was kept as a noninfected, nonmedicated control (NNC). Evaluation of Sensitivity or Resistance Seventeen chicks from each group were weighed individually on the day of inoculation (14 d of age) and then reweighed on d 7 postinoculation (21 d of age). Percentage BW gain between these days was recorded. Five chicks from each group were sacrificed for postmortem examination at 7 d postinoculation (21 d of age). Cecal lesions were scored by the lesion scoring technique described by Johnson and Reid [11]. An oocyst index (0 to 5) was determined by microscopic examination of scrapings from the ceca of chicks sacrificed for lesion scoring at d 7 postinoculation [12]. Mortality was recorded throughout the experimental period, and the exact cause of mortality was confirmed by postmortem examination.
Abbas et al.: EIMERIA TENELLA RESISTANCE Stephen et al. [13] developed the global index (GI), calculated on the basis of weight gain (%), feed conversion (g/g), lesion scores, oocyst index, and mortality (%), by using the formula GI = %WGNNC − [(FM − FNNC) × 10] − (OIM − OIINC) − [(LSM − LSINC) × 2] − (%mortality/2), where GI is the global index, WG is weight gain, F is the FCR, OI is the oocyst index, LS is the lesion score, M is the medicated group, NNC is the noninfected, nonmedicated control group, and INC is the infected, nonmedicated control group. The drug resistance of each isolate of E. tenella against the respective anticoccidial drug was also determined by calculating the GI. In addition, the GI for each test group was calculated as a percentage of the GI for the NNC. The following 5 categories were used for testing resistance to anticoccidials: 1) very good efficacy, ≥90% GINNC; 2) good efficacy, 80 to 89% GINNC; 3) limited efficacy, 70 to 79% GINNC; 4) partially resistant, 50 to 69% GINNC; and 5) resistant, <50% GINNC. Statistical Analysis Data obtained on various parameters were analyzed by ANOVA, and the mean values were compared by Tukey’s test. The experimental unit was the bird. The means for each isolate were compared separately. The results were recorded as mean ± SEM, and the differences among group means were considered significant at P < 0.05.
RESULTS AND DISCUSSION Comparative results of the different parameters used to calculate the GI of salinomycin, maduramicin, and clopidol are summarized in Table 1. It is evident from this table that the average weight gain of the salinomycin-medicated groups did not differ significantly (P > 0.05) from the infected, nonmedicated control groups (INC), except in salinomycin-medicated isolate 1 (SAL 1). The average weight gain of the maduramicin-medicated groups was significantly (P < 0.05) greater than that of the infected, nonmedicated control groups (INC), except in maduramicin-medicated isolate 3 (MAD 3). However, the average weight gain of the clopidol-medicated groups was significantly
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(P < 0.05) greater than that of the respective infected, nonmedicated control groups (INC) for all the isolates. Although the clopidol-medicated groups tended to have greater weight gain in all 3 isolates compared with salinomycin, it was only significant in isolate 3 (CLOP 3), in which the maximum weight gain was found. The results of FCR (Table 1) revealed a pattern relatively similar to that of weight gain among the different isolates of E. tenella. Although a statistical comparison could not be made because of group feeding, the FCR values of medicated groups were numerically lower compared with the infected, nonmedicated groups. The lesion scores of the maduramicin- and clopidol-medicated groups for isolates 1 and 2 differed significantly (P < 0.05) from the lesion scores of the respective infected, nonmedicated groups, but all other medicated groups showed lesion scores similar (P > 0.05) to the respective infected, nonmedicated groups. The results of oocyst scores (Table 1) indicated a significant anticoccidial effect of the salinomycin, maduramicin, and clopidol in isolates 1 and 3 of E. tenella. The oocyst scores calculated for these 2 isolates were lower (P < 0.05) compared with their respective infected, nonmedicated groups. However, isolate 2 showed no difference (P > 0.05) in oocyst scores between the medicated and infected, nonmedicated groups for all the anticoccidials. Mortality (Table 1) in all the medicated groups was greatest in the salinomycin-medicated groups, followed by the maduramicin-medicated groups and the clopidol-medicated groups. However, the mortality percentage was greatest in the infected, nonmedicated groups compared with the medicated groups. Data on the considered criterion (i.e., GI) for the medicated groups (Table 2) compared with the negative and positive controls revealed that the 3 isolates of E. tenella included in the present study had the same level of susceptibility to salinomycin. All the isolates showed partial resistance against salinomycin. However, the isolates of E. tenella showed varying levels of susceptibility to maduramicin and clopidol. Isolate 2 was the most susceptible, followed by isolates 3 and 1, respectively, for maduramicin. Clopidol showed good efficacy against isolates 2 and 3, but isolate 1 showed partial resistance. It was
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Table 1. Comparative values of the mean (and SEM) weight gain, FCR, lesion score, oocyst index, and mortality percentage1
Group
2
SAL 1 MAD 1 CLOP 1 INC 1 SAL 2 MAD 2 CLOP 2 INC 2 SAL 3 MAD 3 CLOP 3 INC 3 NNC SEM
Weight gain (g) 199d 208cd 217cd 154e 212cd 253bc 248bc 174de 221cd 228bcd 264ab 195d 290a 13.83
Weight gain of NNC (%)
FCR3 (g/g)
Lesion score
Oocyst score
Mortality3 (%)
70 71 77 52 75 89 87 62 77 80 93 70 100 —
1.29 1.27 1.27 1.42 1.26 1.20 1.21 1.38 1.23 1.25 1.17 1.32 1.14 —
2.8abc 1.8bc 1.8bc 3.4a 2.4abc 1.6c 1.4c 3.2ab 2.6abc 1.6c 1.6c 3.0abc — 0.43
2.6bc 1.6cd 1.8bcd 4.6a 2.0bcd 2.2bcd 2.2bcd 3.0b 2.2bcd 1.4cd 2.6bc 4.2a — 0.40
13.63 9.09 9.09 22.72 9.09 4.54 4.54 18.18 9.09 9.09 4.54 13.63 0 —
a–e
Means sharing similar superscripts within a column do not differ (P < 0.05). Bird served as the experimental unit. 2 SAL 1 = salinomycin-medicated isolate 1; SAL 2 = salinomycin-medicated isolate 2; SAL 3 = salinomycin-medicated isolate 3; MAD 1 = maduramicin-medicated isolate 1; MAD 2 = maduramicin-medicated isolate 2; MAD 3 = maduramicin-medicated isolate 3; CLOP 1 = clopidol-medicated isolate 1; CLOP 2 = clopidol-medicated isolate 2; CLOP 3 = clopidol-medicated isolate 3; INC 1, 2, and 3 = infected, nonmedicated controls; NNC = noninfected, nonmedicated control. 3 Statistical analysis was not possible because of group feeding of chicks. 1
evident from the results (Table 2) that none of the E. tenella field isolates showed complete sensitivity or complete resistance to the 3 anticoccidials. However, isolate 1 showed cross-resistance between salinomycin and maduramicin (polyether ionophore antibiotics) and multiple resistance among salinomycin, maduramicin, and clopidol. Numerous anticoccidial drugs have been introduced since the end of the 1940s, and over time, resistance to all these anticoccidial drugs has developed in Eimeria species [14, 15]. Several reports are available regarding the development of resistance or loss of sensitivity to the anticoccidial drugs used in the present studies, that is, resistance to salinomycin [13, 16–20], maduramicin [13, 20–22], and clopidol [23]. Therefore, resistance against commonly used anticoccidials is a global issue. Although there are also an increasing number of reports on resistant Eimeria field isolates in countries near to Pakistan, such as India [19, 23–25] and China [17, 20, 26], this is the first study reported in Pakistan. Different formulae have been used by different researchers to find out the sensitivity or resistance to anticoccidial drugs. The older formu-
lae or indices used for determining sensitivity or resistance were the performance index [27] and the anticoccidial index [28–30]. In them, no importance was given to FCR, which is considered to be an important parameter in determining resistance or sensitivity to any anticoccidial drug, because feed costs constitute some 70% of the cost of producing broiler chickens [31]. Presently, the newly devised method of Stephen et al. [13] was used for calculating the GI to detect resistance to anticoccidials in Eimeria spp. In this formula, all 5 parameters—weight gain, FCR, lesion scores, oocyst scores, and mortality—have been given their due importance. The results obtained with this formula corresponded very well to clinical symptoms and postmortem findings. Therefore, the GI can be considered a suitable tool to describe anticoccidial resistance as the above-mentioned conclusive overall picture. Many researchers [13, 19, 32] have correlated the resistance results with practical field conditions with great success by using the GI. Similar to using different parameters to assess the resistance or sensitivity to anticoccidial drugs, using more than 1 field isolate of the Eimeria species is also necessary, because only 1 isolate of Eimeria species is insufficient
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Table 2. Global index and efficacy status of salinomycin, maduramicin, and clopidol against Eimeria tenella field isolates Group1
Global index2
Global index of NNC (%)
Efficacy status3
SAL 1 MAD 1 CLOP 1 SAL 2 MAD 2 CLOP 2 SAL 3 MAD 3 CLOP 3 INC 1 INC 2 INC 3 NNC
64.79 71.36 77.06 71.76 90.13 88.43 74.26 79.96 94.73 37.84 50.41 61.39 110.3
58.73 64.69 69.86 65.05 81.71 80.17 67.32 72.49 85.88 34.30 45.70 55.65 100
4 4 4 4 2 2 4 3 2 — — — —
1
SAL 1 = salinomycin-medicated isolate 1; SAL 2 = salinomycin-medicated isolate 2; SAL 3 = salinomycin-medicated isolate 3; MAD 1 = maduramicin-medicated isolate 1; MAD 2 = maduramicin-medicated isolate 2; MAD 3 = maduramicin-medicated isolate 3; CLOP 1 = clopidol-medicated isolate 1; CLOP 2 = clopidol-medicated isolate 2; CLOP 3 = clopidol-medicated isolate 3; INC 1, 2, and 3 = infected nonmedicated controls; NNC = noninfected, nonmedicated control. 2 Global index (GI) = %WGNNC − [(FM − FNNC) × 10] − (OIM − OIINC) − [(LSM − LSINC) × 2] − (%mortality/2), where WG is weight gain, F is the FCR, OI is the oocyst index, LS is the lesion score, M is the medicated group, NNC is the noninfected, nonmedicated control group, and INC is the infected nonmedicated control group. 3 Efficacy status was calculated as percentage of the GI for the NNC. The following 5 categories were used for testing resistance to anticoccidials: 1) very good efficacy, ≥90% GINNC; 2) good efficacy, 80 to 89% GINNC; 3) limited efficacy, 70 to 79% GINNC; 4) partially resistant, 50 to 69% GINNC; and 5) resistant, <50% GINNC.
to give a conclusive summary of the resistance under field conditions. The Eimeria field isolates of one species consist of several strains. Environmental selection pressures in different geographical locations, as well as the histories of drug use, may differ; therefore, strains resistant in one area may be sensitive in other areas. These strain variants may express different epitopes, leading to immunological diversity, as evidenced by a lack of cross-protection [33]. The resistance test thus gives an overview of the combined effects of all these strains. Sensitive, partially resistant, and resistant strains contribute to this conclusive picture. Therefore, it is not necessary for every resistant strain to have all resistances detected in the isolate [13]. In the present study, a relatively similar pattern was observed, in that all 3 field isolates of E. tenella showed partial resistance against only salinomycin, but in all other treated groups, all the isolates responded differently in terms of sensitivity against maduramicin and clopidol. None of the Eimeria isolates was found to have complete resistance, and none was fully sensitive to the 3 anticoccidials.
Results of the present study revealed partial resistance in all 3 isolates of E. tenella against salinomycin, but all the isolates showed varying degrees of sensitivity to maduramicin and clopidol, except isolate 1, which showed partial resistance against both maduramicin and clopidol. Previous works have also demonstrated the sensitivity of Eimeria isolates to maduramicin [34, 35]. The presence of resistance to salinomycin in all isolates may be due to the extensive use of this ionophore by the poultry feed mills, because this is the cheapest anticoccidial available in Pakistan [36]. Numerous reports [37–40] have also suggested that some Eimeria field isolates may show reduced sensitivity to the polyether ionophore anticoccidials, which have been used extensively since their discovery in 1971. In Pakistan, prophylaxis for coccidiosis is chiefly dependent on the selection of anticoccidial by the feed mills, and single-drug usage programs practice the use of anticoccidials. However, some feed manufacturing companies do not declare the type and quantity of anticoccidial used. Poultry farmers treat coccidiosis by using anticoccidial drugs in the drink-
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366 ing water and also use some anticoccidial feed premix. The medication may continue for 3 to 4 wk. In this way, there is prolonged medication with underdosing of anticoccidial drugs. Furthermore, poultry farmers do not have mixers for the proper mixing of anticoccidials in the feed. Therefore, the other possible reason may be the use of lower dose levels in poultry feed by feed manufacturers and improper mixing by the farmers. The reason for the absence of resistance to the ionophore maduramicin may be that these compounds have not been in use for long periods in Pakistan. Another reason might be the one suggested by Ruff and Danforth [41], that the particular mode of action of ionophores does not allow an easy emergence of resistance against this class of anticoccidials, and only a limited number of cases of complete resistance have been cited in the scientific literature. The partial resistance, or a tendency for the development of resistance, against ionophores in the current study may therefore be attributed to their use for more than a decade, which might have resulted in a gradual reduction in the sensitivity of coccidia. In addition, however, the possibility of cross-resistance among ionophores cannot be ruled out in view of their similar mode of action through the general mechanisms of altering ion transport and disrupting osmotic balance. The present results on ionophores substantiate the findings of Raether and Paeffgen [42], who acknowledged a high degree of cross-resistance between salinomycin and maduramicin. Yet Bedrnik et al. [43] found that maduramicin was effective in controlling isolates of Eimeria that were resistant to other ionophores. Therefore, the difference in the activity of the 2 ionophores (salinomycin and maduramicin) observed in this study may be due to the development of incomplete cross-resistance. For these reasons, forming generalizations about cross-resistance in the polyether ionophores is difficult and imprecise. Isolate 1 was also found to be partially resistant to the chemical anticoccidial clopidol. This loss of sensitivity of isolate 1 to both the ionophores and the chemical anticoccidial clopidol may be due to the development of multiple resistance, because genetic recombination is probably one mechanism by which resistance to
several drugs with different modes of action can arise in the field, as also reviewed by different researchers [15]. Because several reports have shown the presence of resistance in coccidia against ionophores, it might be questioned why these ionophores are still widely used worldwide. First, these anticoccidials have beneficial effects, such as the control of certain gram-positive bacteria. Second, these anticoccidials do not completely suppress the development of coccidia; resistance is usually partial and therefore results in the acquisition of natural immunity by the infected birds [43]. The development of partial resistance to ionophores and clopidol in E. tenella field isolates is also evident from our findings. However, detection of drug resistance in experimental trials does not mean that resistance occurs under field conditions; it only suggests a need to change the routinely used coccidiosis control programs to prolong the life span of anticoccidials, as also suggested by Peeters et al. [44]. The rotation (the use of different anticoccidial drugs among flocks) and shuttle programs (the common use of 2 or more drugs within a single flock) are routinely used to manage the resistance problem. The rotation of anticoccidials with the vaccine containing the anticoccidial drug-sensitive strain has been reported to enhance the sensitivity of Eimeria isolates from 25 to 100% [45]. In a similar way, diclazuril in shuttle programs with other chemical anticoccidials and ionophores is highly efficacious against different Eimeria spp. [46].
CONCLUSIONS AND APPLICATIONS
1. The straight use (single-drug use) of the studied anticoccidials—particularly salinomycin, but also the other 2 drugs, maduramicin and clopidol—should be minimized. 2. These anticoccidials should be used by rotating them with different anticoccidial drugs among flocks. 3. The other possible way to delay the development of resistance may be through the use of shuttle programs.
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