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Studies of resistance to anticoccidials in Eimeria field isolates and pure Eimeria strains
veterinary parasitology ELSEVIER
Veterinary Parasitology69 (1997) 19-29
Studies of resistance to anticoccidials in Eimeria field isolates and pure Eimeria strains B. Stephan a,*, M. Rommel a, A. Daugschies a, A. Haberkorn b Institute of Parasitology, School of Veterinary Medicine, B~nteweg 17, D-30559 Hannot'er, Germany b Bayer AG, Business Group Animal Health, Institute for Parasitology, D-51368 Let,erkusen, Germany
Received 31 May 1996;accepted 13 August 1996
Abstract
Ten Eimeria field isolates from North Germany were studied in battery tests for sensitivity to selected anticoccidials. A high percentage of the Eimeria field isolates (9 out of 10) showed resistance to anticoccidials, mostly multiple resistance. Partial or complete resistance to maduramicin was found in 7 field isolates, to monensin in 6, to salinomycin in 5, to nicarbazin in 8, to halofuginone in 7, to robenidine and toltrazuril in 1, and to diclazuril in 2 field isolates. Multiple resistance had developed in 7 of the 10 isolates. Cross-resistance between maduramicin, monensin, and salinomycin occurred in 5 Eimeria isolates. One isolate showed cross-resistance between diclazuril and toltrazuril. From the resistant isolates 15 pure E. acervulina and 5 pure E. brunetti strains were obtained by single oocyst infections. Seven of the E. acenlulina and 4 of the E. brunetti strains showed resistance or partial resistance that was also present in the original isolate. Ten of 11 resistant strains were multiply resistant. Kevwords: Eimeria spp.; Control methods-Protozoa; Drug resistance
1. I n t r o d u c t i o n
With its ubiquitous distribution, coccidiosis is still one of the main problems of modem broiler production. Without the administration of anticoccidials in feed or in drinking water, economic broiler production is inconceivable. At least US $450 million are spent annually on anticoccidials (Lloyd-Evans, 1991), which illustrates the enormous economic importance of coccidiosis,
* Corresponding author.
0304-4017/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved. PII S0304-40 17(96)01 096-5
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Since the end of the 1940s new anticoccidials have been introduced in increasing numbers, although sooner or later resistance to them develops. A quote from Schnitzer and Grunberg, 1957 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, and other strategies for coccidiosis control, such as the search for safe vaccines, are consequently gaining in importance. In the present study coccidia field isolates from North Germany were investigated for resistance to selected anticoccidials. Pure Eimeria strains were produced from resistant isolates by single oocyst infections, in which one or several resistances are genetically established. These strains should serve as a reference for the development of new anticoccidial active ingredients and as the starting material for investigating the genetic basis of anticoccidial resistance.
2. Materials and methods Ten Eimeria mixed field isolates were used for the resistance studies. Each isolate comes from a different broiler farm in North Germany. All farms were selected by cooperating poultry veterinarians and showed incidence of coccidiosis. Therefore they are not representative of the general coccidiosis situation on German broiler farms. Three farms (isolates 3, 5, 9) are located in the Hannover area, 3 farms (isolates 2, 8, 10) in the Magdeburg area, 2 farms (isolates 6, 7) in the Cuxhaven area and 2 farms (isolates I, 4, 11) in the Oldenburg area. Nothing is known about the use of anticoccidials on these farms. Isolate 11, which came from the same farm as isolate 1, was used only for obtaining Eimeria strains. The individual isolates contained the following species (differentiated on the basis of oocyst morphology, site of colonization, pathology, and clinical signs): isolate 1: E. acervulina, E. mitis; isolate 2: E. acervulina, E. tenella; isolate 3: E. acervulina, E. tenella; isolate 4: E. acervulina, E. brunetti, E. mitis; isolate 5: E. maxima, E. tenella; isolate 6: E. acervulina, E. maxima, E. mitis; isolate 7: E. acervulina, E. brunetti, E. mitis; isolate 8: E. acervulina, E. brunetti, E. mitis, E. tenella; isolate 9: E. acervulina, E. maxima, E. mitis; isolate 10: E. acervulina, E. mitis; isolate 11: E. acervulina, E. mitis. Before performing resistance studies the isolates were passaged through male LSL chicks for multiplication. They were tested as mixed isolates, the infection dose being established after a dose titration. It was 5 × 105 sporulated oocysts for isolates 1, 6, 9, and 10; 105 sporulated oocysts for isolates 2, 5, and 8; 7.5 X 104 sporulated oocysts for isolate 3; and 6 × 104 sporulated oocysts for isolates 4 and 7. For the test of each of the 10 isolates, the anticoccidials maduramicin (5 ppm), monensin (100 ppm), salinomycin (60 ppm), halofuginone (3 ppm), nicarbazin (125 ppm), robenidine (33 ppm), and diclazuril (1 ppm) were used continuously in the feed and toltrazuril (25 ppm) in the drinking water on days 2 and 3 after infection. One group served as non-infected and non-medicated controls (NNC), and another as infected and non-medicated controls (INC). Each group included 10 male LSL chicks 10 days old, which were killed and autopsied 7 days after infection. The following parameters were determined: weight gain
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21
(%), feed conversion (g/g), lesion scoring (Johnson and Reid, 1970), a semiquantitative oocyst index (Hilbrich, 1978), and mortality (%). To calculate a global resistance index from these five parameters, the following formula was developed: GI = %WGNNc -- [(F~ - FNNC) X 10] -- (OI G -- OIINC) -- [(PI G - PIINC) )< 2] -
-
(%mortality/2 ),
where GI is the global index, OI the oocyst index, WG the weight gain, PI the gross-pathological index, F the feed conversion, G the treatment group, NNC the non-infected/non-medicated control and INC the infected/non-medicated control. The formula developed for evaluating resistance studies includes the percentage weight gain as the most economically important parameter, with the largest weighting. Since the differences in feed conversion are often in the decimal range, they are multiplied by 10 to give this equally economically important point appropriate weighting. Oocyst excretion is less important in the assessment of resistance (Reid, 1975) and is therefore evaluated without multiplication. The severity of intestinal leasions is not necessarily correlated with weight gain (Conway et al., 1990), but the difference in the gross-pathological indices is multiplied by 2, since intestinal lesions are a prerequisite for the economically damaging pathophysiological alterations. The percentage mortality is given a high weighting because high mortality due to coccidiosis can be attributed only to insufficient drug efficacy and is the most reliable sign of anticoccidial resistance for the highly pathogenic species E. tenella and E. necatrix (Bedmik, 1983). The global index for each test group was given as a percentage of the global index for the NNC. The following 5 categories were used to assess the efficacy of the tested anticoccidials very good efficacy good efficacy limited efficacy partially resistant resistant
90% GINNc 80% GINNc 70%GINN c 50% GINNc < 50% GINNC
The decision whether all or only some of the species of an isolate were resistant was based on observations during the test evaluation, for example on the absence of typical intestinal lesions or the failure to detect oocysts in swabs. The technique described by Salisch (1985) was modified to obtain pure Eimeria strains. Long cuboids (4 mm high and 4 mm wide) are cut with a scalpel blade from 10% gelatin, which was allowed to solidify in a Petri dish before. 0.5% agar is simultaneously cooled to 38°C and the oocysts are mixed in, so that their count is about 1000/ml. A small amount of this agar-oocyst suspension is transferred with a glass Pasteur pipette to the surface of the gelatin cuboids, in a way that a thin and narrow agar strip is obtained. At a concentration of 1000/ml many oocysts are situated individually and are easily visible in the agar strip. Under microscopic control a small block containing an individual sporulated oocyst is cut out the gelatin cuboid with a scalpel. After subsequent microscopic check that the oocysts are situated individually, such a gelatin block is packed in a commercial gelatin capsule and introduced into the crop of a
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B. Stephan et al. / Veterinary. Parasitology 69 (1997) 1 9 - 2 9
1 0 - d a y - o l d chick. T h e f a e c e s e x c r e t e d o n the 6th d a y a f t e r i n f e c t i o n are c o l l e c t e d a n d i n v e s t i g a t e d f o r o o c y s t s , t o g e t h e r w i t h the i n t e s t i n a l c o n t e n t s o f the r e s p e c t i v e chick, k i l l e d 7 d a y s a f t e r infection. All i n s t r u m e n t s are sterilized, a n d the c h i c k s are k e p t in i s o l a t o r cages. In this m e t h o d a t t e n t i o n m u s t b e p a i d to the age o f the oocysts, w h i c h s h o u l d b e n o m o r e t h a n 2 m o n t h s , since i n f e c t i v i t y a n d viability o f the o o c y s t s d e c r e a s e m a r k e d l y o n storage. E a c h strain o b t a i n e d w a s p a s s a g e d t h r o u g h m a l e L S L c h i c k s for m u l t i p l i c a t i o n a n d a f t e r w a r d s t e s t e d f o r the a n t i c o c c i d i a l r e s i s t a n c e s that were also p r e s e n t in the initial field isolate.
3. Results T h e results o f t h e i n d i v i d u a l e x p e r i m e n t s are s h o w n in T a b l e 1, b r o k e n d o w n b y species a n d a n t i c o c c i d i a l s . T h e G I s o f the i n d i v i d u a l s t u d y g r o u p s are g i v e n as p e r c e n t a g e s o f the c o r r e s p o n d i n g G I for the N N C . All a n t i c o c c i d i a l d r u g s w e r e tested o n e a c h o f the isolates, b u t o n l y r e s i s t a n c e a n d partial r e s i s t a n c e are s h o w n in the table. In the c a s e o f isolate 2 the G I s for the g r o u p s t r e a t e d w i t h m o n e n s i n , m a d u r a m i c i n , s a l i n o m y c i n , h a l o f u g i n o n e , a n d n i c a r b a z i n w e r e n e g a t i v e , s i n c e the m o r t a l i t y rates w e r e b e t w e e n 5 0 a n d 7 0 % . F o r isolate 3 the m o r t a l i t y rates w e r e 10% ( m o n e n s i n , h a l o f u g i n o n e ) , 5 0 % ( m a d u r a m i c i n , s a l i n o m y c i n ) , o r 100% ( n i c a r b a z i n ) . T h e G I s for m a d u r a m icin a n d n i c a r b a z i n w e r e t h e r e f o r e n e g a t i v e . T e n p e r c e n t o f the c h i c k s treated with Eimeria
Table 1 Individual results of the resistance experiments with Eimeria field isolates 1 to 10 Isolate
Eimeria species
MAD
MON
SAL
l 2 3 4 5 6 6 77 8 9 10
acv + mit ten ten acv + bru + mit max + ten max acv + mit acv + bru + mit acv + bru + mit + ten acv + max + mit acv + mit
36 GI-20 GI-9 59
GI-38 49
GI-23
47
35 48 48
52
43
ll
51
45
69
69
6
NIC
HAL
ROB
GI-29 GI - 5 1 62 48 34 34
GI-20 35 51 21 GI - 2 4 GI - 24
13
31 67 59
4 65
TOL
DIC
45
60
55
MAD = maduramicin, MON = monensin, SAL = salinomycin, NIC = nicarbazin, HAL = halofuginone, ROB = robenidine, TOL = toltrazuril, DIC = diclazuril. acv = E. acervulina, bru = E. brunetti, max = E. maxima, mit = E. mitis, ten = E. tenella, GI = global index. Printed in bold = resistant, normally printed = partially resistant. All the anticoccidials were tested on each of the isolates. Only resistance and partial resistance are listed in the table. The GI values of the empty cells are greater than 70% of the GI of the NNC and therefore belonging to the categories very good, good or limited efficacy. According to the formula shown on page 4, the Gls for some groups of the isolates two, three and six were negative due to high mortality or very poor feed conversion, and could therefore not be expressed as percentages.
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B. Stephan et al. / Veterina~ Parasitology 69 (1997) 19-29 Table 2 Frequencies of resistances against anticoccidials in Eimeria field isolates
Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate
l 2 3 4 5 6 7 8 9 10
MAD
MON
SAL
NIC
HAL
• • • C)
• •
• •
•
• •
©
• • 0 • •
• • C) • •
•
•
O
•
•
0
©
• © 0
ROB
TOL
DIC
O
©
0
: resistant. O = partial resistant. For further explanation see Table 1. •
halofuginone died in the experiment with isolate 6, and the GI for this group was negative because of the additionally very poor feed conversion. The negative GIs cannot be expressed as percentages and are therefore listed separately in the table. For a better overview the resistance experiments with the Eimeria field isolates are summarized once again in Table 2. The frequency of partial or full resistance to the individual anticoccidials is shown. The table shows clearly that isolates 2, 3, 4, 5, 6, 8, and 10 are multiply resistant. Moreover, isolates 2, 3, 6, 8, and 10 showed cross-resistance between the ionophore antibiotics maduramicin, monensin, and salinomycin. In isolate 10 there was also a partial cross-resistance between diclazuril and toltrazuril. Isolates 1, 2, 3, 4, 5, 6, 8, 10 and 11 were used to obtain pure Eimeria strains. Two E. acervulina strains ( l / l a c v , l / 2 a c v ) were obtained from isolate 1, three E. acervulina strains ( 2 / l a c y , 2/2acv, 2/3acv) from isolate 2, one E. acervulina strain
Table 3 Resistance of the Eimeria strains isolated by single ©©cystinfection
Eimeria strain 2 / 3 acv 4/2bru 4/3bru 6 / I acv 8/lbru 8/2bru 8/3bru 10/lacy 10/2acv 10/3acv 10/4acv
MAD
MON
SAL
NIC
HAL 57 42
38 65 53 23 37
62 52 GI - 1 8 46 44
GI - 9 57 52
For further explanation see Table 1.
45 51 31 GI - 7 68
GI - 48
39
ROB
TOL
DIC
5 67 54
57 34 62 46
33
61 62 69 G1 - 2 1 1 50 GI -40 43 36
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B. Stephan et al. / VeterinaryParasitology69 (1997) 19-29
Table 4 Comparison of resistance patterns of Eimeria strains, isolated by single oocyst infection, with those of the initial isolates Eimeria strain
Resistancesof the initial isolate
Resistances of the strain
2/3acv 4/2bru 4/3acv 6/lacv 8/lbru 8/2bru 8/3bru 10/lacv 10/2acv 10/3acv 10/4acv
MAD, MON, SAL, NIC, HAL MAD, NIC, HAL MAD, NIC, HAL MON, NIC, HAL, ROB MAD, MON, SAL, NIC, HAL MAD, MON, SAL, NIC, HAL MAD, MON, SAL, NIC, HAL MAD, MON, SAL, NIC, HAL TOL, DIC MAD, MON, SAL, NIC, HAL TOL, DIC MAD, MON, SAL, NIC, HAL TOL, DIC MAD, MON, SAL, NIC, HAL TOL, DIC
NIC NIC, HAL MAD, HAL MON, HAL MON, SAL, HAL MAD, MON, SAL, NIC, HAL MON, SAL, HAL MAD,DIC MAD,MON, SAL, NIC, ItAL TOL, DIC MAD,MON, SAL, HAL, TOL, DIC MAD,MON, HAL, TOL, DIC
Printed in bold = resistant. Normally printed = partial resistant. For further explanation see Table 1. (3/lacv)
from isolate 3, two E. brunetti strains ( 4 / l b r u , 4 / 2 b r u ) and one E. acervulina strain ( 4 / 3 a c v ) from isolate 4, one E. aeervulina strain ( 6 / l a c v ) from isolate 6, three E. brunetti strains ( 8 / l b r u , 8 / 2 b r u , 8 / 3 b r u ) from isolate 8, four E. acervulina strains ( 1 0 / l a c v , 10/2acv, 10/3acv, 1 0 / 4 a c v ) from isolate 10, and three E. acervulina strains ( 1 1 / l a c v , 11/2acv, 1 1 / 3 acv) from isolate 11. With 4 chicks used for each isolate (2 for isolate 3) the strain yield was thus 52.6%. Despite the use of 8 chicks, the experiments with isolate 5 were unsuccessful. Table 3 shows the resistance of the Eimeria strains. For some strains from isolates 8 and 10 the GIs for some groups were negative because of very poor weight gain and poor feed conversion. For these the percentages cannot be calculated. All other values are the GIs of the respective groups, expressed as percentages of the GI of the corresponding NNC. Eleven of the 20 Eimeria strains (55%) had resistances that were also present in the initial isolate. In Table 4 the initial isolates are compared with the resistant strains obtained from them. There were no strains in the experiments that had resistances other than those present in the initial isolate.
4. Discussion We must first ask how resistance to anticoccidials should be defined under practical conditions, and what importance should be attached to the individual parameters in resistance studies. Bedrnfk (1983) has established that a reduced efficacy of an anticoccidial always has an adverse effect on weight gain a n d / o r on the feed conversion. These factors play a decisive part in practice, since they determine the economic success of the operation.
B. Stephan et al. / Veterinary Parasitology 69 (1997) 19-29
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Oocyst excretion alone is insufficient to assess the efficacy of anticoccidials, and indeed it is misleading (Reid, 1975). Many publications have shown that oocyst production correlates poorly with weight development, with intestinal lesions, and in the highly pathogenic species E. tenella and E. necatrix even with mortality (Engle et al., 1967; Norton and Joyner, 1968; Waletzky, 1970; Barwick, 1973; Greuel et al., 1978; Chapman, 1979; Grant et al., 1986; Weber and Frigg, 1986). No anticoccidial can completely suppress oocyst production under practical conditions (Jeffers, 1978). An example is isolate 7. All groups treated with anticoccidials gained more weight than the NNC, but apart from robenidine none of the products prevented excretion of oocysts. On the other hand, the growth-promoting effect of some anticoccidials certainly cannot be neglected. This applies in particular to the ionophore antibiotics (Reid et al., 1972; Clarke et al., 1974; Frigg and Schramm, 1977; Chappel and Babcock, 1979; Migaki et al., 1979; Jeffers et al., 1988; Seikh et al., 1988), robenidine (Reid et al., 1970), and toltrazuril (Kutzer and Leibetseder, 1985; Kutzer et al., 1985). It is therefore conceivable that this growth-promoting action masks the effects of an Eimeria infection in comparison with other anticoccidials. For Eimeria strains with a low resistance the experimental results can therefore be misinterpreted, since in spite of a high oocyst index, for example, very good overall results can be obtained because of the growth promotion. The gross-pathological index, which is based on intestinal lesions, is also not necessarily correlated with weight development of the chicks (Conway et al., 1990), as demonstrated by the chicks treated with toltrazuril from the experiment with isolate 1, the nicarbazin group from isolate 7, and the monensin group from isolate 9. In all these groups high intestinal lesion indices were observed side by side with excellent weight gains. The occurrence of deaths due to coccidiosis is the most reliable sign of resistance to anticoccidials (Bedrnlk, 1983), and for the highly pathogenic species E. tenella and E. necatrix it is therefore a very good indicator of resistance problems in the field. However, other causes of death such as diseases and stress must be excluded, so that they are not falsely assessed as a sign of resistance. The answer to our initial question is therefore that anticoccidial resistance under practical conditions is present when a deteriorating weight gain and feed conversion together with oocyst excretion, intestinal lesions, and mortality give a conclusive overall picture. We must once again mention here the necessity of standardizing study procedures, as called for by McDougald (1993), since only in this way comparable results can be expected about resistance to anticoccidials, its distribution, and the efficacy of new anticoccidial products. Ten-day-old chicks were used in the resistance tests, and were given relatively high infection doses. We did not take into account the fact that under practical conditions an increasing infection pressure first builds up gradually, and does not always lead to clinically manifest coccidiosis. Up to this time the chicks may already have developed immunity or partial immunity, so that the effects of the Eimeria infection may be less pronounced than in the laboratory test. Further, this test lasts only 7 days, so that long-term effects such as compensatory growth and reinfections are not taken into account (Bedrnlk, 1983). Because of all these differences from field conditions, labora-
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B. Stephan et al. / Veterinary Parasitology 69 (1997) 19-29
tory results can be extrapolated to the situation in farms only with certain limitations (G~isslein, 1990). A formula was developed to calculate a global resistance index from the five parameters investigated. The results obtained with this formula are corresponding very well to clinical symptoms and post-mortem findings. Therefore the formula can be considered a suitable tool to describe anticoccidial resistance as the above mentioned conclusive overall picture. Many anticoccidial drugs have been introduced since the end of the 1940s and sooner or later resistance to all these products developed in Eimeria species (Chapman, 1993). A lot of papers were published, reporting on resistance to the anticoccidials used in our studies, i.e. resistance to halofuginone (Mathis and McDougald, 1982; Hamet, 1986; G~isslein, 1990), to nicarbazin (Jeffers, 1974a; Jeffers, 1974b; Mathis and McDougald, 1982; Fuller and McDougald, 1986; Chapman, 1989a; Katae et al., 1989; Zhao et al., 1990), to robenidine (Jeffers, 1974b; Mathis and McDougald, 1982; Hamet, 1986; Fanyao et al., 1994), to the ionophore antibiotics (Raether and Paeffgen, 1989; Katae et al., 1989; Chapman, 1989b; Chapman and Shirley, 1989; Ziomko et al., 1992; Fanyao et al., 1994), to diclazuril (Kawazoe and Di Fabio, 1993; Haberkorn, 1994) and to toltrazuril (Haberkorn, 1994). There was an increasing number of reports on multiply resistant Eimeria field isolates during the last few years (Chapman, 1989b; Chapman, 1993; Haberkorn, 1993; Peeters and Geeroms, 1993; Haberkorn, 1994). The results of our studies are very similar to the findings of the authors quoted above. There are several advantages to the above-described method of single oocyst infection. Packing the gelatin block carrying an oocyst into gelatin capsules allows a more reliable introduction of the oocyst into the test animal. In contrast, if gelatin oocyst suspensions are placed directly onto slides, cutting out of oocysts and the subsequent control are more difficult. If individual oocysts in water droplets are taken up in a syringe, they often remain stuck to the internal wall of the syringe and the inoculation is unsuccessful. Further advantages are the easy recognition, marking, and isolation of the oocysts. It is easy to carry out a subsequent microscopic examination. The method is thus distinguished by reliability and simplicity and is not too time-consuming: 4 - 6 oocysts can be isolated in 30 min. In our studies 20 strains were obtained from 38 chicks, a success rate of 52.6%. Of the 20 pure Eimeria strains, eleven (55%) had a partial or complete resistance. The method of individual oocyst infection is therefore well suited for obtaining resistant strains from resistant field isolates. No studies have yet been done on the recovery of sensitivity of pure anticoccidial-resistant Eimeria strains in the absence of the respective product or by medication with a different and chemically unrelated anticoccidial product. For the strains obtained here it can be assumed that the resistance will be permanent, owing to genetic fixation. However, to be completely sure, and to induce higher levels of resistance, in future the strains should be passaged under anticoccidial medication. The Eimeria field isolates consist mostly of several species and a large number of strains. A resistance test thus gives an overview of the combined effects of all these strains. Resistant, partly resistant, and sensitive strains contribute to this overall picture. This is why not every resistant strain needs to have all resistances detected in the isolate. What rather tends to happen is that each strain can develop resistances, partial
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27
resistances, and sensitivity to anticoccidials in entirely free combinations. Therefore, only in one strain isolated by single oocyst infection (10/2acv) all resistances of the initial isolate were detected, while all other Eimeria strains had only some of the resistances found in the field isolate. In conclusion, a high percentage of the Eimeria field isolates (9 out of 10) showed resistance to anticoccidials, mostly a multiple resistance. Resistance to anticoccidials is therefore a widespread phenomenon, which even occurs with the modem triazine derivatives. From the resistant isolates 20 pure strains were obtained, eleven of which had resistances of their starting isolates. The described method of individual oocyst infection is well suited for the isolation of resistant strains from field isolates resistant to anticoccidials. References Barwick, M.W., 1973. An investigation of methods of expressing the coccidiostat intake of chickens with reference to clopidol. Proc. 4th Eur. Poult. Conf., London, pp. 151-156. Bedrn~, P., 1983. Evaluation of sensitivity of coccidia to ionophores, Arch. Gefliigelk., 47: 129-133. Chapman, H.D., 1979. Studies on the sensitivity of recent field isolates of Eimeria maxima to monensin. Avian Pathol., 8: 181-186. Chapman, H.D., 1989a, Sensitivity of field isolates of Eimeria tenella to anticoccidial drugs in the chicken. Res. Vet. Sci., 47: 125-128. Chapman, H.D., 1989b. Field isolates of Eimeria tenella: sensitivity to diclazuril, maduramicin, narasin, salinomycin and a mixture of nicarbazin/narasin. In: P. Yvor~ (Editor), Coccidia and Intestinal Coccidiomorphs. INRA Publ., Paris, pp. 323-326. Chapman, H.D., 1993. Resistance to anticoccidial drugs in fowl. Parasitol. Today, 9: 159-162. Chapman, H.D. and Shirley, M.W., 1989. Sensitivity of field isolates of Eimeria species to monensin and lasalocid in the chicken. Res. Vet. Sci., 46:114-117. Chappel, L.R. and Babcock, W.E., 1979. Field trials comparing salinomycin (Coxistac), monensin and lasalocid in the control of coccidiosis in broilers. Poult. Sci., 58: 304-307. Clarke, M.L., Diaz, M., Guilloteau, B., Hudd, D.L. and Stoker, J.W., 1974. European field evaluation of monensin, a new anticoccidial agent. Avian Pathol., 3: 25-35. Conway, D.P., McKenzie, M.E, and Dayton, A.D., 1990. Relationship of coccidial lesion scores and weight gain in infections of Eimeria acert,ulina, E. maxima and E. tenella in broilers. Avian Pathol., 19: 489-496. Engle, A.T., Humphrey, R.P, and Johnson, C.A., 1967. Buquinolate, a new broad-spectrum coccidiostat. Poult. Sci., 46: 810-818. Fanyao, K., Changshen, N. and Peiyun, Y., 1994. A survey of drug resistance to coccidiostats of 15 field isolates of Eimeria tenella in China. In: M. Xie (Editor), Proc. 2nd Asian Conf. on Coccidiosis. Guangdong Academy of Agricultural Sciences, Guangzhou, China, p. 219. Frigg, M. and Schramm, H., 1977. Comparative anticoccidial activity of lasalocid sodium (Avatec) in chicks. Efficacy against European strains of coccidia. Arch. Gefliigelk., 4 1 : 3 1 - 3 4 . Fuller, A.L. and McDougald, L.R., 1986. Epidemiology and anticoccidial sensitivity of coccidia strains isolated from broiler farms in the US: a survey of 99 field isolates. In: L.R. McDougald, L,P. Joyner and P.L. Long (Editors), Research in Avian Coccidiosis. Univ. Georgia, Atlanta, pp. 348-352. G~isslein, U., 1990. Untersuchungen zur Sensitivitiit von Kokzidienisolaten aus Gefliigelmastbetrieben gegen ausgesuchte Antikokzidia im Feld- und Laborversuch. Diss.. Tier~irztliche Hochschule, Hannover, Germany. Grant, R.J., King, H.F. and Myers, G.H., 1986. The effect on broiler growth and coccidiosis parameters of feeding halofuginone and monensin continuously in the same commercial pens for two years. In: L.R. McDougald, L.P. Joyner and P.L. Long (Editors), Research in Avian Coccidiosis. Univ. Georgia, Atlanta, pp. 431-441.
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Greuel, E., Braunius, W.W. and Ktihnhold, W., 1978. Zur Wirksamkeit verchiedener Coccidiostatica gegen Eimeria Feldisolate. Arch. Gefliigelk., 42: 16-22. Haberkorn, A., 1993. Studies on cross resistance and compatibility of toltrazuril with other anticoccidials. In: J.R. Barta and M.A. Fernando (Editors), Proc. Vlth Intern. Coccidiosis Conf. Univ. of Guelph, Guelph, Ont., Canada, p. 153. Haberkorn, A., 1994. Investigations on crossresistance of coccidia against toltrazuril and diclazuril. In: N. Suzuki (Editor), 9th Japanese German Cooperative Symposium On Protozoan Diseases. Obihiro University, Obihiro, Hokkaido, Japan, p. 51. Hamet, N,, 1986. Resistance to anticoccidial drugs in poultry farms in France from 1975 to 1984. In: L.R. McDougald, L.P. Joyner and P.L. Long (Editors), Research in Avian Coccidiosis. Univ. Georgia, Atlanta, pp. 415-420. Hilbrich, P., 1978. Krankheiten des GeflUgels unter besonderer Beriicksichtigung der Haltung und Ftitterung. Hermann Kuhn KG, Schwenningen am Neckar, Germany. Jeffers, T.K., 1974a. Eimeria tenella: incidence, distribution, and anticoccidial drug resistance of isolants in major broiler-producing areas. Avian Dis., 18: 74-84. Jeffers, T.K., 1974b. E. acervulina and E. maxima: incidence and anticoccidial drug resistance of isolants in major broiler-producing areas. Avian Dis., 18:331-342. Jeffers, T.K., 1978. Genetics of coccidia and the host response. In: P.L. Long, K.N. Boorman and B.M. Freeman (Editors), Avian Coccidiosis. Brit. Poult. Sci. Ltd, Edinburgh, pp. 50-125. Jeffers, T.K., Tonkison, L.V. and Callender, M.E., 1988. Anticoccidial efficacy of narasin in battery cage trials. Poult. Sci., 67: 1043-1049. Johnson, J. and Reid, W.M., 1970. Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens. Exp. Parasitol., 28: 30-36. Katae, H., Nakai, M., Yoshida, M. and Spelman, J., 1989. Efficacy of currently used anticoccidials against Eimeria isolated over a 4 year period from poultry farms in Japan. In: P. Yvor6 (Editor), Coccidia and Intestinal Coccidiomorphs. INRA Publ., Paris, pp. 377-382. Kawazoe, U. and Di Fabio, J., 1993. Sensitivity of field isolates of Eimeria species to diclazuril. In: J.R. Barta and M.A. Fernando (Editors), Proc. VIth Intern. Coccidiosis Conf., Univ. of Guelph, Guelph, Ont., Canada, p. 153. Kutzer, E. and Leibetseder, J., 1985. Untersuchungen iiber die Wirkung eines neuen Antikokzidiums (Bay Vi 9142) bei Broilern. Wien. Tier'~irztl. Mschr., 72: 321-330. Kutzer, E., Freiler, I., Leibetseder, J. and Mitterlehner, A., 1985. Untersuchungen fiber die Wirkung eines neuen Antikokzidiums (Bay Vi 9142) bei Broilern. II. Wien. Tier'~ztl. Mschr., 72: 330-334. Lloyd-Evans, L.P.M., 1991. Markets for anticoccidials. In: L.P.M. Lloyd-Evans (Editor), Animal Pharm, Trends in Veterinary Research and Development. PJB Publications, Richmond, UK, p. 35. Mathis, G.F. and McDougald, L.R., 1982. Drug responsiveness of field isolates of chicken coccidia. Poult. Sci., 61: 38-45. McDougald, L.R., 1993. Chemotherapy of coccidiosis. In: J.R. Barta and M.A. Fernando (Editors), Proc. VItb Intern. Coccidiosis Conf., Univ. of Guelph, Guelph, Ont., Canada, pp. 45-47. Migaki, T.T., Chappel, L.R. and Babcock, W.E., 1979. Anticoccidial efficacy of a new polyether antibiotic, salinomycin, in comparison to monensin and lasalocid in battery trials. Poult. Sci., 58:1192-1196. Norton, C.C. and Joyner, L.P., 1968. Coccidiostatic action of meticlorpindol. Vet. Rec., 83: 317-323. Peeters, J.E. and Geeroms, R., 1993. Sensitivity of avian coccidia from Belgian broiler farms to six chemical and five ionophore anticoccidials. In: J.R. Barta and M.A. Fernando (Editors), Proc. VIth Intern. Coccidiosis Conf., Univ. of Guelph, Guelph, Ont., Canada, p. 154. Raether, W. and Paeffgen, E., 1989. Drug sensitivity of coccidia recently selected from broiler farms in Europe. In: P. Yvor~ (Editor), Coccidia and Intestinal Coccidiomorphs. INRA Publ., Pads, pp. 309-312. Reid, W.M., 1975. Relative value of oocyst counts in evaluating anticoccidial activity. Avian Dis., 19: 802-811. Reid, W.M., Kowalski, L. and Rice, J., 1972. Anticoccidial activity of monensin in floor-pen experiments. Poult. Sci., 51: 139-146. Reid, W.M., Kowalski, L., Taylor, E.M. and Johnson, J., 1970. Efficacy evaluations of robenzidene for control of coccidiosis in chickens. Avian Dis., 14: 788-796. Salisch, H., 1985. Beitrag zur in vitro Kultur von Eimeria tenella: die Entwicklung eines durch Einzeloozys-
B. Stephan et aL / Veterinary Parasitology 69 (1997) 19-29
29
teninfektion gewonnenen Stammes in Eikulturen, in Kiikennierenzellen und in einer homologen Zellinie. Diss., Tier~irztliche Hochschule, Hannover, Germany. Schnitzer, R.J. and Grunberg, E., 1957. Drug Resistance of Microorganisms. Academic Press, New York. Seikh, G.N., Verma, B.B. and Thakur, D.K., 1988. Chemoprophylactic and chemotherapeutic efficacy of lasalocid sodium (Avatec l~) in experimental intestinal coccidiosis of poultry. Indian Vet. J., 65: 779-782. Waletzky, E,, 1970, Laboratory anticoccidial evaluation trials: review of designs, variables, criteria, and predictive value for field use, Exp. Parasitol., 28: 42-62. Weber, G.M. and Frigg, M., 1986. Comparative efficacy of lasalocid and monensin against coccidia of two field isolates, repeatedly propagated in medicated chicks. In: L.R. McDougald, P.L. Joyner and L.P. Long (Editors), Research in Avian Coccidiosis. Univ. Georgia, Atlanta, pp. 363-371. Zhao, S.Y., Yu, H., Duan, J.S. and Shen, H., 1990. The effectiveness of some anticoccidial drugs against Eimeria acerL,ulina. Chin. J. Vet. Med., 16: 29-30. Ziomko, I., Kucz~nska, E. and Cencek, T., 1992. Opornosc kokcydii Eimeria tenella na kokcydiostatyki stosowane aktualnie w hodowli drobiu (Resistance of Eimeria tenella to coccidiostats used in poultry husbandry). Medycyna Weterynaryjna, 48: 255-257.
Veterinary Parasitology69 (1997) 19-29
Studies of resistance to anticoccidials in Eimeria field isolates and pure Eimeria strains B. Stephan a,*, M. Rommel a, A. Daugschies a, A. Haberkorn b Institute of Parasitology, School of Veterinary Medicine, B~nteweg 17, D-30559 Hannot'er, Germany b Bayer AG, Business Group Animal Health, Institute for Parasitology, D-51368 Let,erkusen, Germany
Received 31 May 1996;accepted 13 August 1996
Abstract
Ten Eimeria field isolates from North Germany were studied in battery tests for sensitivity to selected anticoccidials. A high percentage of the Eimeria field isolates (9 out of 10) showed resistance to anticoccidials, mostly multiple resistance. Partial or complete resistance to maduramicin was found in 7 field isolates, to monensin in 6, to salinomycin in 5, to nicarbazin in 8, to halofuginone in 7, to robenidine and toltrazuril in 1, and to diclazuril in 2 field isolates. Multiple resistance had developed in 7 of the 10 isolates. Cross-resistance between maduramicin, monensin, and salinomycin occurred in 5 Eimeria isolates. One isolate showed cross-resistance between diclazuril and toltrazuril. From the resistant isolates 15 pure E. acervulina and 5 pure E. brunetti strains were obtained by single oocyst infections. Seven of the E. acenlulina and 4 of the E. brunetti strains showed resistance or partial resistance that was also present in the original isolate. Ten of 11 resistant strains were multiply resistant. Kevwords: Eimeria spp.; Control methods-Protozoa; Drug resistance
1. I n t r o d u c t i o n
With its ubiquitous distribution, coccidiosis is still one of the main problems of modem broiler production. Without the administration of anticoccidials in feed or in drinking water, economic broiler production is inconceivable. At least US $450 million are spent annually on anticoccidials (Lloyd-Evans, 1991), which illustrates the enormous economic importance of coccidiosis,
* Corresponding author.
0304-4017/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved. PII S0304-40 17(96)01 096-5
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B. Stephan et al. / Veterinary Parasitology 69 (1997) 19-29
Since the end of the 1940s new anticoccidials have been introduced in increasing numbers, although sooner or later resistance to them develops. A quote from Schnitzer and Grunberg, 1957 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, and other strategies for coccidiosis control, such as the search for safe vaccines, are consequently gaining in importance. In the present study coccidia field isolates from North Germany were investigated for resistance to selected anticoccidials. Pure Eimeria strains were produced from resistant isolates by single oocyst infections, in which one or several resistances are genetically established. These strains should serve as a reference for the development of new anticoccidial active ingredients and as the starting material for investigating the genetic basis of anticoccidial resistance.
2. Materials and methods Ten Eimeria mixed field isolates were used for the resistance studies. Each isolate comes from a different broiler farm in North Germany. All farms were selected by cooperating poultry veterinarians and showed incidence of coccidiosis. Therefore they are not representative of the general coccidiosis situation on German broiler farms. Three farms (isolates 3, 5, 9) are located in the Hannover area, 3 farms (isolates 2, 8, 10) in the Magdeburg area, 2 farms (isolates 6, 7) in the Cuxhaven area and 2 farms (isolates I, 4, 11) in the Oldenburg area. Nothing is known about the use of anticoccidials on these farms. Isolate 11, which came from the same farm as isolate 1, was used only for obtaining Eimeria strains. The individual isolates contained the following species (differentiated on the basis of oocyst morphology, site of colonization, pathology, and clinical signs): isolate 1: E. acervulina, E. mitis; isolate 2: E. acervulina, E. tenella; isolate 3: E. acervulina, E. tenella; isolate 4: E. acervulina, E. brunetti, E. mitis; isolate 5: E. maxima, E. tenella; isolate 6: E. acervulina, E. maxima, E. mitis; isolate 7: E. acervulina, E. brunetti, E. mitis; isolate 8: E. acervulina, E. brunetti, E. mitis, E. tenella; isolate 9: E. acervulina, E. maxima, E. mitis; isolate 10: E. acervulina, E. mitis; isolate 11: E. acervulina, E. mitis. Before performing resistance studies the isolates were passaged through male LSL chicks for multiplication. They were tested as mixed isolates, the infection dose being established after a dose titration. It was 5 × 105 sporulated oocysts for isolates 1, 6, 9, and 10; 105 sporulated oocysts for isolates 2, 5, and 8; 7.5 X 104 sporulated oocysts for isolate 3; and 6 × 104 sporulated oocysts for isolates 4 and 7. For the test of each of the 10 isolates, the anticoccidials maduramicin (5 ppm), monensin (100 ppm), salinomycin (60 ppm), halofuginone (3 ppm), nicarbazin (125 ppm), robenidine (33 ppm), and diclazuril (1 ppm) were used continuously in the feed and toltrazuril (25 ppm) in the drinking water on days 2 and 3 after infection. One group served as non-infected and non-medicated controls (NNC), and another as infected and non-medicated controls (INC). Each group included 10 male LSL chicks 10 days old, which were killed and autopsied 7 days after infection. The following parameters were determined: weight gain
B. Stephan et al. / Veterinary Parasitology 69 (1997) 19-29
21
(%), feed conversion (g/g), lesion scoring (Johnson and Reid, 1970), a semiquantitative oocyst index (Hilbrich, 1978), and mortality (%). To calculate a global resistance index from these five parameters, the following formula was developed: GI = %WGNNc -- [(F~ - FNNC) X 10] -- (OI G -- OIINC) -- [(PI G - PIINC) )< 2] -
-
(%mortality/2 ),
where GI is the global index, OI the oocyst index, WG the weight gain, PI the gross-pathological index, F the feed conversion, G the treatment group, NNC the non-infected/non-medicated control and INC the infected/non-medicated control. The formula developed for evaluating resistance studies includes the percentage weight gain as the most economically important parameter, with the largest weighting. Since the differences in feed conversion are often in the decimal range, they are multiplied by 10 to give this equally economically important point appropriate weighting. Oocyst excretion is less important in the assessment of resistance (Reid, 1975) and is therefore evaluated without multiplication. The severity of intestinal leasions is not necessarily correlated with weight gain (Conway et al., 1990), but the difference in the gross-pathological indices is multiplied by 2, since intestinal lesions are a prerequisite for the economically damaging pathophysiological alterations. The percentage mortality is given a high weighting because high mortality due to coccidiosis can be attributed only to insufficient drug efficacy and is the most reliable sign of anticoccidial resistance for the highly pathogenic species E. tenella and E. necatrix (Bedmik, 1983). The global index for each test group was given as a percentage of the global index for the NNC. The following 5 categories were used to assess the efficacy of the tested anticoccidials very good efficacy good efficacy limited efficacy partially resistant resistant
90% GINNc 80% GINNc 70%GINN c 50% GINNc < 50% GINNC
The decision whether all or only some of the species of an isolate were resistant was based on observations during the test evaluation, for example on the absence of typical intestinal lesions or the failure to detect oocysts in swabs. The technique described by Salisch (1985) was modified to obtain pure Eimeria strains. Long cuboids (4 mm high and 4 mm wide) are cut with a scalpel blade from 10% gelatin, which was allowed to solidify in a Petri dish before. 0.5% agar is simultaneously cooled to 38°C and the oocysts are mixed in, so that their count is about 1000/ml. A small amount of this agar-oocyst suspension is transferred with a glass Pasteur pipette to the surface of the gelatin cuboids, in a way that a thin and narrow agar strip is obtained. At a concentration of 1000/ml many oocysts are situated individually and are easily visible in the agar strip. Under microscopic control a small block containing an individual sporulated oocyst is cut out the gelatin cuboid with a scalpel. After subsequent microscopic check that the oocysts are situated individually, such a gelatin block is packed in a commercial gelatin capsule and introduced into the crop of a
22
B. Stephan et al. / Veterinary. Parasitology 69 (1997) 1 9 - 2 9
1 0 - d a y - o l d chick. T h e f a e c e s e x c r e t e d o n the 6th d a y a f t e r i n f e c t i o n are c o l l e c t e d a n d i n v e s t i g a t e d f o r o o c y s t s , t o g e t h e r w i t h the i n t e s t i n a l c o n t e n t s o f the r e s p e c t i v e chick, k i l l e d 7 d a y s a f t e r infection. All i n s t r u m e n t s are sterilized, a n d the c h i c k s are k e p t in i s o l a t o r cages. In this m e t h o d a t t e n t i o n m u s t b e p a i d to the age o f the oocysts, w h i c h s h o u l d b e n o m o r e t h a n 2 m o n t h s , since i n f e c t i v i t y a n d viability o f the o o c y s t s d e c r e a s e m a r k e d l y o n storage. E a c h strain o b t a i n e d w a s p a s s a g e d t h r o u g h m a l e L S L c h i c k s for m u l t i p l i c a t i o n a n d a f t e r w a r d s t e s t e d f o r the a n t i c o c c i d i a l r e s i s t a n c e s that were also p r e s e n t in the initial field isolate.
3. Results T h e results o f t h e i n d i v i d u a l e x p e r i m e n t s are s h o w n in T a b l e 1, b r o k e n d o w n b y species a n d a n t i c o c c i d i a l s . T h e G I s o f the i n d i v i d u a l s t u d y g r o u p s are g i v e n as p e r c e n t a g e s o f the c o r r e s p o n d i n g G I for the N N C . All a n t i c o c c i d i a l d r u g s w e r e tested o n e a c h o f the isolates, b u t o n l y r e s i s t a n c e a n d partial r e s i s t a n c e are s h o w n in the table. In the c a s e o f isolate 2 the G I s for the g r o u p s t r e a t e d w i t h m o n e n s i n , m a d u r a m i c i n , s a l i n o m y c i n , h a l o f u g i n o n e , a n d n i c a r b a z i n w e r e n e g a t i v e , s i n c e the m o r t a l i t y rates w e r e b e t w e e n 5 0 a n d 7 0 % . F o r isolate 3 the m o r t a l i t y rates w e r e 10% ( m o n e n s i n , h a l o f u g i n o n e ) , 5 0 % ( m a d u r a m i c i n , s a l i n o m y c i n ) , o r 100% ( n i c a r b a z i n ) . T h e G I s for m a d u r a m icin a n d n i c a r b a z i n w e r e t h e r e f o r e n e g a t i v e . T e n p e r c e n t o f the c h i c k s treated with Eimeria
Table 1 Individual results of the resistance experiments with Eimeria field isolates 1 to 10 Isolate
Eimeria species
MAD
MON
SAL
l 2 3 4 5 6 6 77 8 9 10
acv + mit ten ten acv + bru + mit max + ten max acv + mit acv + bru + mit acv + bru + mit + ten acv + max + mit acv + mit
36 GI-20 GI-9 59
GI-38 49
GI-23
47
35 48 48
52
43
ll
51
45
69
69
6
NIC
HAL
ROB
GI-29 GI - 5 1 62 48 34 34
GI-20 35 51 21 GI - 2 4 GI - 24
13
31 67 59
4 65
TOL
DIC
45
60
55
MAD = maduramicin, MON = monensin, SAL = salinomycin, NIC = nicarbazin, HAL = halofuginone, ROB = robenidine, TOL = toltrazuril, DIC = diclazuril. acv = E. acervulina, bru = E. brunetti, max = E. maxima, mit = E. mitis, ten = E. tenella, GI = global index. Printed in bold = resistant, normally printed = partially resistant. All the anticoccidials were tested on each of the isolates. Only resistance and partial resistance are listed in the table. The GI values of the empty cells are greater than 70% of the GI of the NNC and therefore belonging to the categories very good, good or limited efficacy. According to the formula shown on page 4, the Gls for some groups of the isolates two, three and six were negative due to high mortality or very poor feed conversion, and could therefore not be expressed as percentages.
23
B. Stephan et al. / Veterina~ Parasitology 69 (1997) 19-29 Table 2 Frequencies of resistances against anticoccidials in Eimeria field isolates
Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate Isolate
l 2 3 4 5 6 7 8 9 10
MAD
MON
SAL
NIC
HAL
• • • C)
• •
• •
•
• •
©
• • 0 • •
• • C) • •
•
•
O
•
•
0
©
• © 0
ROB
TOL
DIC
O
©
0
: resistant. O = partial resistant. For further explanation see Table 1. •
halofuginone died in the experiment with isolate 6, and the GI for this group was negative because of the additionally very poor feed conversion. The negative GIs cannot be expressed as percentages and are therefore listed separately in the table. For a better overview the resistance experiments with the Eimeria field isolates are summarized once again in Table 2. The frequency of partial or full resistance to the individual anticoccidials is shown. The table shows clearly that isolates 2, 3, 4, 5, 6, 8, and 10 are multiply resistant. Moreover, isolates 2, 3, 6, 8, and 10 showed cross-resistance between the ionophore antibiotics maduramicin, monensin, and salinomycin. In isolate 10 there was also a partial cross-resistance between diclazuril and toltrazuril. Isolates 1, 2, 3, 4, 5, 6, 8, 10 and 11 were used to obtain pure Eimeria strains. Two E. acervulina strains ( l / l a c v , l / 2 a c v ) were obtained from isolate 1, three E. acervulina strains ( 2 / l a c y , 2/2acv, 2/3acv) from isolate 2, one E. acervulina strain
Table 3 Resistance of the Eimeria strains isolated by single ©©cystinfection
Eimeria strain 2 / 3 acv 4/2bru 4/3bru 6 / I acv 8/lbru 8/2bru 8/3bru 10/lacy 10/2acv 10/3acv 10/4acv
MAD
MON
SAL
NIC
HAL 57 42
38 65 53 23 37
62 52 GI - 1 8 46 44
GI - 9 57 52
For further explanation see Table 1.
45 51 31 GI - 7 68
GI - 48
39
ROB
TOL
DIC
5 67 54
57 34 62 46
33
61 62 69 G1 - 2 1 1 50 GI -40 43 36
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B. Stephan et al. / VeterinaryParasitology69 (1997) 19-29
Table 4 Comparison of resistance patterns of Eimeria strains, isolated by single oocyst infection, with those of the initial isolates Eimeria strain
Resistancesof the initial isolate
Resistances of the strain
2/3acv 4/2bru 4/3acv 6/lacv 8/lbru 8/2bru 8/3bru 10/lacv 10/2acv 10/3acv 10/4acv
MAD, MON, SAL, NIC, HAL MAD, NIC, HAL MAD, NIC, HAL MON, NIC, HAL, ROB MAD, MON, SAL, NIC, HAL MAD, MON, SAL, NIC, HAL MAD, MON, SAL, NIC, HAL MAD, MON, SAL, NIC, HAL TOL, DIC MAD, MON, SAL, NIC, HAL TOL, DIC MAD, MON, SAL, NIC, HAL TOL, DIC MAD, MON, SAL, NIC, HAL TOL, DIC
NIC NIC, HAL MAD, HAL MON, HAL MON, SAL, HAL MAD, MON, SAL, NIC, HAL MON, SAL, HAL MAD,DIC MAD,MON, SAL, NIC, ItAL TOL, DIC MAD,MON, SAL, HAL, TOL, DIC MAD,MON, HAL, TOL, DIC
Printed in bold = resistant. Normally printed = partial resistant. For further explanation see Table 1. (3/lacv)
from isolate 3, two E. brunetti strains ( 4 / l b r u , 4 / 2 b r u ) and one E. acervulina strain ( 4 / 3 a c v ) from isolate 4, one E. aeervulina strain ( 6 / l a c v ) from isolate 6, three E. brunetti strains ( 8 / l b r u , 8 / 2 b r u , 8 / 3 b r u ) from isolate 8, four E. acervulina strains ( 1 0 / l a c v , 10/2acv, 10/3acv, 1 0 / 4 a c v ) from isolate 10, and three E. acervulina strains ( 1 1 / l a c v , 11/2acv, 1 1 / 3 acv) from isolate 11. With 4 chicks used for each isolate (2 for isolate 3) the strain yield was thus 52.6%. Despite the use of 8 chicks, the experiments with isolate 5 were unsuccessful. Table 3 shows the resistance of the Eimeria strains. For some strains from isolates 8 and 10 the GIs for some groups were negative because of very poor weight gain and poor feed conversion. For these the percentages cannot be calculated. All other values are the GIs of the respective groups, expressed as percentages of the GI of the corresponding NNC. Eleven of the 20 Eimeria strains (55%) had resistances that were also present in the initial isolate. In Table 4 the initial isolates are compared with the resistant strains obtained from them. There were no strains in the experiments that had resistances other than those present in the initial isolate.
4. Discussion We must first ask how resistance to anticoccidials should be defined under practical conditions, and what importance should be attached to the individual parameters in resistance studies. Bedrnfk (1983) has established that a reduced efficacy of an anticoccidial always has an adverse effect on weight gain a n d / o r on the feed conversion. These factors play a decisive part in practice, since they determine the economic success of the operation.
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25
Oocyst excretion alone is insufficient to assess the efficacy of anticoccidials, and indeed it is misleading (Reid, 1975). Many publications have shown that oocyst production correlates poorly with weight development, with intestinal lesions, and in the highly pathogenic species E. tenella and E. necatrix even with mortality (Engle et al., 1967; Norton and Joyner, 1968; Waletzky, 1970; Barwick, 1973; Greuel et al., 1978; Chapman, 1979; Grant et al., 1986; Weber and Frigg, 1986). No anticoccidial can completely suppress oocyst production under practical conditions (Jeffers, 1978). An example is isolate 7. All groups treated with anticoccidials gained more weight than the NNC, but apart from robenidine none of the products prevented excretion of oocysts. On the other hand, the growth-promoting effect of some anticoccidials certainly cannot be neglected. This applies in particular to the ionophore antibiotics (Reid et al., 1972; Clarke et al., 1974; Frigg and Schramm, 1977; Chappel and Babcock, 1979; Migaki et al., 1979; Jeffers et al., 1988; Seikh et al., 1988), robenidine (Reid et al., 1970), and toltrazuril (Kutzer and Leibetseder, 1985; Kutzer et al., 1985). It is therefore conceivable that this growth-promoting action masks the effects of an Eimeria infection in comparison with other anticoccidials. For Eimeria strains with a low resistance the experimental results can therefore be misinterpreted, since in spite of a high oocyst index, for example, very good overall results can be obtained because of the growth promotion. The gross-pathological index, which is based on intestinal lesions, is also not necessarily correlated with weight development of the chicks (Conway et al., 1990), as demonstrated by the chicks treated with toltrazuril from the experiment with isolate 1, the nicarbazin group from isolate 7, and the monensin group from isolate 9. In all these groups high intestinal lesion indices were observed side by side with excellent weight gains. The occurrence of deaths due to coccidiosis is the most reliable sign of resistance to anticoccidials (Bedrnlk, 1983), and for the highly pathogenic species E. tenella and E. necatrix it is therefore a very good indicator of resistance problems in the field. However, other causes of death such as diseases and stress must be excluded, so that they are not falsely assessed as a sign of resistance. The answer to our initial question is therefore that anticoccidial resistance under practical conditions is present when a deteriorating weight gain and feed conversion together with oocyst excretion, intestinal lesions, and mortality give a conclusive overall picture. We must once again mention here the necessity of standardizing study procedures, as called for by McDougald (1993), since only in this way comparable results can be expected about resistance to anticoccidials, its distribution, and the efficacy of new anticoccidial products. Ten-day-old chicks were used in the resistance tests, and were given relatively high infection doses. We did not take into account the fact that under practical conditions an increasing infection pressure first builds up gradually, and does not always lead to clinically manifest coccidiosis. Up to this time the chicks may already have developed immunity or partial immunity, so that the effects of the Eimeria infection may be less pronounced than in the laboratory test. Further, this test lasts only 7 days, so that long-term effects such as compensatory growth and reinfections are not taken into account (Bedrnlk, 1983). Because of all these differences from field conditions, labora-
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tory results can be extrapolated to the situation in farms only with certain limitations (G~isslein, 1990). A formula was developed to calculate a global resistance index from the five parameters investigated. The results obtained with this formula are corresponding very well to clinical symptoms and post-mortem findings. Therefore the formula can be considered a suitable tool to describe anticoccidial resistance as the above mentioned conclusive overall picture. Many anticoccidial drugs have been introduced since the end of the 1940s and sooner or later resistance to all these products developed in Eimeria species (Chapman, 1993). A lot of papers were published, reporting on resistance to the anticoccidials used in our studies, i.e. resistance to halofuginone (Mathis and McDougald, 1982; Hamet, 1986; G~isslein, 1990), to nicarbazin (Jeffers, 1974a; Jeffers, 1974b; Mathis and McDougald, 1982; Fuller and McDougald, 1986; Chapman, 1989a; Katae et al., 1989; Zhao et al., 1990), to robenidine (Jeffers, 1974b; Mathis and McDougald, 1982; Hamet, 1986; Fanyao et al., 1994), to the ionophore antibiotics (Raether and Paeffgen, 1989; Katae et al., 1989; Chapman, 1989b; Chapman and Shirley, 1989; Ziomko et al., 1992; Fanyao et al., 1994), to diclazuril (Kawazoe and Di Fabio, 1993; Haberkorn, 1994) and to toltrazuril (Haberkorn, 1994). There was an increasing number of reports on multiply resistant Eimeria field isolates during the last few years (Chapman, 1989b; Chapman, 1993; Haberkorn, 1993; Peeters and Geeroms, 1993; Haberkorn, 1994). The results of our studies are very similar to the findings of the authors quoted above. There are several advantages to the above-described method of single oocyst infection. Packing the gelatin block carrying an oocyst into gelatin capsules allows a more reliable introduction of the oocyst into the test animal. In contrast, if gelatin oocyst suspensions are placed directly onto slides, cutting out of oocysts and the subsequent control are more difficult. If individual oocysts in water droplets are taken up in a syringe, they often remain stuck to the internal wall of the syringe and the inoculation is unsuccessful. Further advantages are the easy recognition, marking, and isolation of the oocysts. It is easy to carry out a subsequent microscopic examination. The method is thus distinguished by reliability and simplicity and is not too time-consuming: 4 - 6 oocysts can be isolated in 30 min. In our studies 20 strains were obtained from 38 chicks, a success rate of 52.6%. Of the 20 pure Eimeria strains, eleven (55%) had a partial or complete resistance. The method of individual oocyst infection is therefore well suited for obtaining resistant strains from resistant field isolates. No studies have yet been done on the recovery of sensitivity of pure anticoccidial-resistant Eimeria strains in the absence of the respective product or by medication with a different and chemically unrelated anticoccidial product. For the strains obtained here it can be assumed that the resistance will be permanent, owing to genetic fixation. However, to be completely sure, and to induce higher levels of resistance, in future the strains should be passaged under anticoccidial medication. The Eimeria field isolates consist mostly of several species and a large number of strains. A resistance test thus gives an overview of the combined effects of all these strains. Resistant, partly resistant, and sensitive strains contribute to this overall picture. This is why not every resistant strain needs to have all resistances detected in the isolate. What rather tends to happen is that each strain can develop resistances, partial
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27
resistances, and sensitivity to anticoccidials in entirely free combinations. Therefore, only in one strain isolated by single oocyst infection (10/2acv) all resistances of the initial isolate were detected, while all other Eimeria strains had only some of the resistances found in the field isolate. In conclusion, a high percentage of the Eimeria field isolates (9 out of 10) showed resistance to anticoccidials, mostly a multiple resistance. Resistance to anticoccidials is therefore a widespread phenomenon, which even occurs with the modem triazine derivatives. From the resistant isolates 20 pure strains were obtained, eleven of which had resistances of their starting isolates. The described method of individual oocyst infection is well suited for the isolation of resistant strains from field isolates resistant to anticoccidials. References Barwick, M.W., 1973. An investigation of methods of expressing the coccidiostat intake of chickens with reference to clopidol. Proc. 4th Eur. Poult. Conf., London, pp. 151-156. Bedrn~, P., 1983. Evaluation of sensitivity of coccidia to ionophores, Arch. Gefliigelk., 47: 129-133. Chapman, H.D., 1979. Studies on the sensitivity of recent field isolates of Eimeria maxima to monensin. Avian Pathol., 8: 181-186. Chapman, H.D., 1989a, Sensitivity of field isolates of Eimeria tenella to anticoccidial drugs in the chicken. Res. Vet. Sci., 47: 125-128. Chapman, H.D., 1989b. Field isolates of Eimeria tenella: sensitivity to diclazuril, maduramicin, narasin, salinomycin and a mixture of nicarbazin/narasin. In: P. Yvor~ (Editor), Coccidia and Intestinal Coccidiomorphs. INRA Publ., Paris, pp. 323-326. Chapman, H.D., 1993. Resistance to anticoccidial drugs in fowl. Parasitol. Today, 9: 159-162. Chapman, H.D. and Shirley, M.W., 1989. Sensitivity of field isolates of Eimeria species to monensin and lasalocid in the chicken. Res. Vet. Sci., 46:114-117. Chappel, L.R. and Babcock, W.E., 1979. Field trials comparing salinomycin (Coxistac), monensin and lasalocid in the control of coccidiosis in broilers. Poult. Sci., 58: 304-307. Clarke, M.L., Diaz, M., Guilloteau, B., Hudd, D.L. and Stoker, J.W., 1974. European field evaluation of monensin, a new anticoccidial agent. Avian Pathol., 3: 25-35. Conway, D.P., McKenzie, M.E, and Dayton, A.D., 1990. Relationship of coccidial lesion scores and weight gain in infections of Eimeria acert,ulina, E. maxima and E. tenella in broilers. Avian Pathol., 19: 489-496. Engle, A.T., Humphrey, R.P, and Johnson, C.A., 1967. Buquinolate, a new broad-spectrum coccidiostat. Poult. Sci., 46: 810-818. Fanyao, K., Changshen, N. and Peiyun, Y., 1994. A survey of drug resistance to coccidiostats of 15 field isolates of Eimeria tenella in China. In: M. Xie (Editor), Proc. 2nd Asian Conf. on Coccidiosis. Guangdong Academy of Agricultural Sciences, Guangzhou, China, p. 219. Frigg, M. and Schramm, H., 1977. Comparative anticoccidial activity of lasalocid sodium (Avatec) in chicks. Efficacy against European strains of coccidia. Arch. Gefliigelk., 4 1 : 3 1 - 3 4 . Fuller, A.L. and McDougald, L.R., 1986. Epidemiology and anticoccidial sensitivity of coccidia strains isolated from broiler farms in the US: a survey of 99 field isolates. In: L.R. McDougald, L,P. Joyner and P.L. Long (Editors), Research in Avian Coccidiosis. Univ. Georgia, Atlanta, pp. 348-352. G~isslein, U., 1990. Untersuchungen zur Sensitivitiit von Kokzidienisolaten aus Gefliigelmastbetrieben gegen ausgesuchte Antikokzidia im Feld- und Laborversuch. Diss.. Tier~irztliche Hochschule, Hannover, Germany. Grant, R.J., King, H.F. and Myers, G.H., 1986. The effect on broiler growth and coccidiosis parameters of feeding halofuginone and monensin continuously in the same commercial pens for two years. In: L.R. McDougald, L.P. Joyner and P.L. Long (Editors), Research in Avian Coccidiosis. Univ. Georgia, Atlanta, pp. 431-441.
28
B. Stephan et al. / Veterinary Parasitology 69 (1997) 19-29
Greuel, E., Braunius, W.W. and Ktihnhold, W., 1978. Zur Wirksamkeit verchiedener Coccidiostatica gegen Eimeria Feldisolate. Arch. Gefliigelk., 42: 16-22. Haberkorn, A., 1993. Studies on cross resistance and compatibility of toltrazuril with other anticoccidials. In: J.R. Barta and M.A. Fernando (Editors), Proc. Vlth Intern. Coccidiosis Conf. Univ. of Guelph, Guelph, Ont., Canada, p. 153. Haberkorn, A., 1994. Investigations on crossresistance of coccidia against toltrazuril and diclazuril. In: N. Suzuki (Editor), 9th Japanese German Cooperative Symposium On Protozoan Diseases. Obihiro University, Obihiro, Hokkaido, Japan, p. 51. Hamet, N,, 1986. Resistance to anticoccidial drugs in poultry farms in France from 1975 to 1984. In: L.R. McDougald, L.P. Joyner and P.L. Long (Editors), Research in Avian Coccidiosis. Univ. Georgia, Atlanta, pp. 415-420. Hilbrich, P., 1978. Krankheiten des GeflUgels unter besonderer Beriicksichtigung der Haltung und Ftitterung. Hermann Kuhn KG, Schwenningen am Neckar, Germany. Jeffers, T.K., 1974a. Eimeria tenella: incidence, distribution, and anticoccidial drug resistance of isolants in major broiler-producing areas. Avian Dis., 18: 74-84. Jeffers, T.K., 1974b. E. acervulina and E. maxima: incidence and anticoccidial drug resistance of isolants in major broiler-producing areas. Avian Dis., 18:331-342. Jeffers, T.K., 1978. Genetics of coccidia and the host response. In: P.L. Long, K.N. Boorman and B.M. Freeman (Editors), Avian Coccidiosis. Brit. Poult. Sci. Ltd, Edinburgh, pp. 50-125. Jeffers, T.K., Tonkison, L.V. and Callender, M.E., 1988. Anticoccidial efficacy of narasin in battery cage trials. Poult. Sci., 67: 1043-1049. Johnson, J. and Reid, W.M., 1970. Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens. Exp. Parasitol., 28: 30-36. Katae, H., Nakai, M., Yoshida, M. and Spelman, J., 1989. Efficacy of currently used anticoccidials against Eimeria isolated over a 4 year period from poultry farms in Japan. In: P. Yvor6 (Editor), Coccidia and Intestinal Coccidiomorphs. INRA Publ., Paris, pp. 377-382. Kawazoe, U. and Di Fabio, J., 1993. Sensitivity of field isolates of Eimeria species to diclazuril. In: J.R. Barta and M.A. Fernando (Editors), Proc. VIth Intern. Coccidiosis Conf., Univ. of Guelph, Guelph, Ont., Canada, p. 153. Kutzer, E. and Leibetseder, J., 1985. Untersuchungen iiber die Wirkung eines neuen Antikokzidiums (Bay Vi 9142) bei Broilern. Wien. Tier'~irztl. Mschr., 72: 321-330. Kutzer, E., Freiler, I., Leibetseder, J. and Mitterlehner, A., 1985. Untersuchungen fiber die Wirkung eines neuen Antikokzidiums (Bay Vi 9142) bei Broilern. II. Wien. Tier'~ztl. Mschr., 72: 330-334. Lloyd-Evans, L.P.M., 1991. Markets for anticoccidials. In: L.P.M. Lloyd-Evans (Editor), Animal Pharm, Trends in Veterinary Research and Development. PJB Publications, Richmond, UK, p. 35. Mathis, G.F. and McDougald, L.R., 1982. Drug responsiveness of field isolates of chicken coccidia. Poult. Sci., 61: 38-45. McDougald, L.R., 1993. Chemotherapy of coccidiosis. In: J.R. Barta and M.A. Fernando (Editors), Proc. VItb Intern. Coccidiosis Conf., Univ. of Guelph, Guelph, Ont., Canada, pp. 45-47. Migaki, T.T., Chappel, L.R. and Babcock, W.E., 1979. Anticoccidial efficacy of a new polyether antibiotic, salinomycin, in comparison to monensin and lasalocid in battery trials. Poult. Sci., 58:1192-1196. Norton, C.C. and Joyner, L.P., 1968. Coccidiostatic action of meticlorpindol. Vet. Rec., 83: 317-323. Peeters, J.E. and Geeroms, R., 1993. Sensitivity of avian coccidia from Belgian broiler farms to six chemical and five ionophore anticoccidials. In: J.R. Barta and M.A. Fernando (Editors), Proc. VIth Intern. Coccidiosis Conf., Univ. of Guelph, Guelph, Ont., Canada, p. 154. Raether, W. and Paeffgen, E., 1989. Drug sensitivity of coccidia recently selected from broiler farms in Europe. In: P. Yvor~ (Editor), Coccidia and Intestinal Coccidiomorphs. INRA Publ., Pads, pp. 309-312. Reid, W.M., 1975. Relative value of oocyst counts in evaluating anticoccidial activity. Avian Dis., 19: 802-811. Reid, W.M., Kowalski, L. and Rice, J., 1972. Anticoccidial activity of monensin in floor-pen experiments. Poult. Sci., 51: 139-146. Reid, W.M., Kowalski, L., Taylor, E.M. and Johnson, J., 1970. Efficacy evaluations of robenzidene for control of coccidiosis in chickens. Avian Dis., 14: 788-796. Salisch, H., 1985. Beitrag zur in vitro Kultur von Eimeria tenella: die Entwicklung eines durch Einzeloozys-
B. Stephan et aL / Veterinary Parasitology 69 (1997) 19-29
29
teninfektion gewonnenen Stammes in Eikulturen, in Kiikennierenzellen und in einer homologen Zellinie. Diss., Tier~irztliche Hochschule, Hannover, Germany. Schnitzer, R.J. and Grunberg, E., 1957. Drug Resistance of Microorganisms. Academic Press, New York. Seikh, G.N., Verma, B.B. and Thakur, D.K., 1988. Chemoprophylactic and chemotherapeutic efficacy of lasalocid sodium (Avatec l~) in experimental intestinal coccidiosis of poultry. Indian Vet. J., 65: 779-782. Waletzky, E,, 1970, Laboratory anticoccidial evaluation trials: review of designs, variables, criteria, and predictive value for field use, Exp. Parasitol., 28: 42-62. Weber, G.M. and Frigg, M., 1986. Comparative efficacy of lasalocid and monensin against coccidia of two field isolates, repeatedly propagated in medicated chicks. In: L.R. McDougald, P.L. Joyner and L.P. Long (Editors), Research in Avian Coccidiosis. Univ. Georgia, Atlanta, pp. 363-371. Zhao, S.Y., Yu, H., Duan, J.S. and Shen, H., 1990. The effectiveness of some anticoccidial drugs against Eimeria acerL,ulina. Chin. J. Vet. Med., 16: 29-30. Ziomko, I., Kucz~nska, E. and Cencek, T., 1992. Opornosc kokcydii Eimeria tenella na kokcydiostatyki stosowane aktualnie w hodowli drobiu (Resistance of Eimeria tenella to coccidiostats used in poultry husbandry). Medycyna Weterynaryjna, 48: 255-257.