Parasitology (1975), 71, 41-49 With 3figuresin the text
41
Eimeria tenella in chickens: development of resistance to quinolone anticoccidial drugs H. D. CHAPMAN Houghton Poultry Research Station, Houghton, Huntingdon, Cambs. (Received 4 December 1974) SUMMARY
The development of drug resistance by the present Houghton strain of Eimeria tenella to the quinolones, methyl benzoquate and buquinolate, was found to take place after a single experimental passage. The development of resistance was independent of drug selection pressure and showed cross resistance to other quinolones, but not to amprolium and robenidine. When the Weybridge, Beltsville and Elberfeld strains of E. tenella were compared under similar laboratory conditions, the Weybridge and Elberfeld strains developed resistance to methyl benzoquate after 6 passages and the Beltsville after 5. Studies on the response of the Houghton strain to methyl benzoquate and buquinolate revealed that the drugs did not completely control the infection as measured by weight gain and that oocyst production was not suppressed. These observations indicate that the strain had already acquired some resistance to these drugs. This was confirmed by examining the resistance to methyl benzoquate of a culture of the Houghton strain of E. tenella which had been stored frozen in liquid nitrogen since 1969. This showed full sensitivity to the drug and developed resistance after 8 passages. This suggests that drug tolerance has been acquired by the Houghton strain since 1969. Oocyst lines were established from the Houghton strain by infecting single birds with approximately 10 oocysts. Eleven of these lines were found to be sensitive to methyl benzoquate, and nine to give rise to resistant parasites. It is concluded that the Houghton strain is contaminated by a small number of resistant oocysts which can be eliminated from a culture by dilution of the challenge inoculum. One of these Houghton oocyst lines, sensitive to methyl benzoquate, developed resistance after 8 serial passages. INTRODUCTION
Several workers have shown the apparent ease with which the coccidia acquire resistance to the quinolones; resistance of Eimeria to buquinolate, decoquinate and methyl benzoquate developing after 4-6 passages in the presence of drug (McLoughlin, 1970; McLoughlin & Chute, 1971, 1973). Field strains of E. tenella resistant to quinolones have been described (Millard, 1970; McManus, Campbell & Cuckler, 1968) and in a recent study of the incidence of drug resistance in the field, Jeffers (1974) found that resistance to decoquinate and buquinolate was present in 40-60% of E. tenella isolates examined. McManus et al. (1968) found that species of Eimeria differ in their rate of development of resistance. In studies
42
H. D. CHAPMAN
with amquinolate resistance developed after 4 passages in E. tenella, E. brunetti and E. maxima and after 8 passages in E. acervulina but no information was given on possible differences between strains of the same species. According to Ryley & Betts (1973), different strains of one species isolated from different localities usually vary in their sensitivity to a drug with which they have not had prior contact. Joyner (1970) has described differences in drug efficacy against the Houghton and Weybridge strains of E. tenella and has suggested that inherent heterogenicity among field strains may influence the development of drug resistance. In this paper, the development of resistance by the Houghton strain of E. tenella to the quinolones, methyl benzoquate and buquinolate, is described. A comparison is made between the Weybridge, Beltsville and Elberfeld strains of this species. The anomalous behaviour of the Houghton strain is discussed and the restoration of sensitivity of this strain to methyl benzoquate is described. METHODS
Animals. Twelve-day-old Light Sussex chickens were used. They were kept in wire floored metal cages and maintained as described by Long (1968). Drugs were incorporated into a standard ration supplemented with vitamin K. The ration was formulated for experimental work with coccidia; details of its composition have been given by Ryley & Betts (1973). The food was prepared in a special mill used only for experimental diets making contamination with anticoccidial drugs extremely unlikely. Infective material. The Houghton strain of E. tenella chosen for study has been maintained in this laboratory by serial passage through chickens. The Elberfeld strain was supplied by Dr A. Haberkorn and a culture of the Houghton strain frozen in liquid nitrogen since 1969 by Dr L. P. Joyner. Graded infective doses of oocysts were achieved by serial dilution of a freshly prepared inoculum which had been accurately counted. All suspensions of oocysts were stirred whilst inoculating the birds to ensure that each received a similar dose. After 7 days the birds were killed, weighed and oocysts were harvested from the caeca using the method described by Long (1972). Oocysts in the faeces of infected groups of birds were counted by methods described by Long & Rowell (1958). Experimental procedure for developing drug resistance. Birds were allocated to groups of ten using a restricted randomization procedure which approximately equalized average initial weights, and inoculated with 100000 oocysts. The number of passages required to develop resistance was then established by serially passaging the coccidia in the presence of sub-optimal concentrations of drug. Birds were infected with 100000 oocysts at each passage. The design of the experiments was based upon procedures established by McLoughlin & Gardiner (1961). The essential feature was the division of the original strain into two oocyst lines, one passaged in the presence of the drug to which resistance is to be developed, the other passaged for the same number of times in the absence of drug. This latter line served as a reference standard,
Resistance of E. tenella to quinolone drugs
43
against which the response of medicated chicks to infection could be measured, and served to ensure ttnat no change in VnruYence accompanied. \3&fi> seqvxence oi passages. The response of this line to drugs after resistance had been developed in the 'resistant' line also enabled a check to be made that the strain had retained its original sensitivity despite the sequence of passages. Both weight gain and oocyst production were used as parameters for measuring drug activity. Weight gains were expressed as a percentage of the weight gain of uninfected, non-medicated birds. The % weight gain of birds infected and non-medicated is also included in the figures. Restoration of sensitivity of the Houghton strain to methyl benzoquate. Twentyfour birds were each given 10 oocysts. The dose of 10 oocysts was only approximate, since attempts to verify the numbers present by direct counting after the method of Joyner & Norton (1973) were unsuccessful. Twenty-one of these chickens became infected and small numbers of oocysts were harvested. Each oocyst line established in this way was then re-passaged in 2 birds to build up the number of oocysts and handled separately throughout the experiment. Strict precautions were taken to ensure that cross contamination did not occur. The response of each line to 40 parts per million (ppm) methyl benzoquate was then established by inoculating groups of 5 birds individually with 100000 oocysts. In some of these lines oocyst production was completely suppressed. Further attempts were made to passage them at lower drug concentrations. In those lines where oocysts were recovered from the primary passage, the degree of resistance was evaluated by passage for a second time in the presence of drug.
RESULTS
Development of resistance by the Houghton strain to methyl benzoquate and buquinolate The response of the Houghton strain to methyl benzoquate is illustrated in Fig. 1 A. Primary passage in the presence of different concentrations of this drug resulted in a dose-response relationship in which maximum activity was found at concentrations of 1 p.p.m. and above. Below this concentration drug activity declined to a level similar to infected non-medicated birds. A similar relationship was found for buquinolate with maximum activity at 32 p.p.m. and above and little activity below 10 p.p.m. (Fig. IB). Oocysts were harvested from birds fed 40 p.p.m. methyl benzoquate and 82.5 p.p.m. buquinolate and the dose-response relationship again established (Fig. 1A, B). All concentrations of drug used failed to control infections and weight gains of medicated groups were similar to infected non-medicated birds. Development of resistance by the Houghton strain passaged at different concentrations of drug The strain was passaged in birds given drug in the food at concentrations ranging from 0-1 to 40 p.p.m. methyl benzoquate and 4-0 to 500 p.p.m. buquinolate. After a single passage, oocysts were harvested from the various lines and the degree of resistance established by measuring the response to 40 p.p.m. methyl
44 1 VU —
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Drug concentration
Fig. 1. Response of the Houghton strain of Eimeria tenella to drugs. (A) Methyl benzoquate. (B) Buquinolate. Key: % weight gain of infected non -medicated birds included in brackets after the passage number. 9 — 0 , primary passage (25%); O—O> 2nd passage (32%). Drug concentrations plotted on a log scale. 100 i -
80
I
60
DO
40
20
I
0-1
I
I
10 10 100 Drug concentration used in passage
1000
Fig. 2. Response of the Houghton strain passaged at different drug concentrations to 40 parts per million (ppm) methyl benzoquate and 82-5 ppm buquinolate. 0 — 0 , methyl benzoquate; O—O> buquinolate. Weight gain of infected, nonmedicated birds was 32 % of non-infected controls. benzoquate and 82-5 p.p.m. buquinolate. Results are illustrated in Fig. 2. In those lines where the drug had initially controlled the infections weight gains were similar to infected non-medicated birds indicating the development of resistance. In those lines where no control of the infection had been found (0-1 p.p.m. methyl benzoquate, 4-0-8-0 p.p.m. buquinolate) resistance did not develop.
Resistance of E. tenella to quinolone drugs
45
Table 1. Effect of drugs on two resistant lines of the Houghton strain of Eimeria tenella and a control line which had been passaged for the same number of times in the absence of drug Weight gain-% uninfected non-medicated birds Drug concentration (ppm) Methyl benzoquate 40 Buquinolate 82-5 Decoquinate 40 Robenidine 33 Amprolium 125 Non-medicated
Methyl benzoquate resistant
Buquinolate resistant
Control
16 35 20 103 93 39
24 42 38 96 98 32
84 81 95 102 91 25
Cross resistance The methyl benzoquate and buquinolate resistant lines were fully cross resistant to each other and to decoquinate but showed no cross resistance to robenidine and amprolium (Table 1). Development of resistance by the Weybridge, Beltsville and Elberfeld strains of E. tenella The response to methyl benzoquate is illustrated in Fig. 3A-C. At concentrations of 10 p.p.m. and above, methyl benzoquate showed greater activity against these strains than had been found for the Houghton strain. Weight gains were approximately 100 % that of uninfected non-medicated birds. Both Beltsville and Elberfeld strains showed weight gains of approximately 80 % at 1 p.p.m. with similar activity at 5 p.p.m. for the Weybridge strain. Declining activity was found at lower drug concentrations. Oocyst production was initially completely suppressed by 10 p.p.m. methyl benzoquate and so the strains were passaged at the maximum concentration of drug which would permit sufficient oocysts to be harvested to continue the passage. After 6 serial passages with the Weybridge and Elberfeld strains and 5 passages with the Beltsville strain, weight gains declined to levels similar to infected non-medicated birds. The dose response relationship was then established at different drug concentrations (Fig. 3A-C). The response of the Elberfeld strain (Fig. 3C) to different concentrations of drug showed more variation than the other strains. In all three strains, complete failure to control infections was found at all drug levels indicating the development of resistance. Oocyst production by the various strains of E. tenella at different concentrations of methyl benzoquate Control of the Houghton strain by methyl benzoquate has been shown to be incomplete as judged by the weight gain of infected medicated birds. This was further studied by examining oocyst production of the strains of E. tenella in birds given different concentrations of drug (Table 2). Oocyst production by the Weybridge, Beltsville and Elberfeld strains was completely suppressed by 10 p.p.m.
46
H. D. CHAPMAN A
100
^
80
E
60
rc
DO
l>40 *
20
0-1
10
10
100 0-1
10 10 Drug concentration
100 0 1
10
10
100
- E 80 cN c
/ 60 -
/
So JZ
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/
rf
^-^°
20 -
01
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10
10
1
10 100 0-1 Drug concentration
10
100
Fig. 3. Response of different strains of Eimeria tenella to methyl benzoquate. (A) Weybridge: 0 — # , primary passage (32%); O—O, 6th passage (24%). (B) Beltsville: # — • , primary passage (21%); O—O, 5th passage (39%). (C) Elberfeld: • — • , primary passage (28%); O—O, 6th passage (23%). (D) Houghton (1969): # — • , primary passage (37%); O—O. 8th passage (31 %). (E) Houghton (sensitive isolate): # — # , primary passage (40%); O—O, 8th passage (22%). Table 2. Oocyst production of different strains of Eimeria tenella in response to different concentrations of methyl benzoquate Oocysts produced (millions/bird) Drug concentration (ppm in food) Strain of E. tenella Houghton (current) Weybridge Beltsville Elberfeld Houghton (1969) Houghton (sensitive isolate)
40
20
10
5
1
0
23-6
17-9
15-7
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
24- 2 16- 1 1- 0 1- 0 0 0
12-1 29-0 10-2 10-2 9-8 39-6
28-8 17-4 24-0 26-6 11-9 29-8
Resistance of E. tenella to quinolone drugs
47
methyl benzoquate, but no control of the Houghton strain was obtained. These results indicated that some resistance had already been acquired by this strain. Development of resistance by the Houghton strain prepared from material frozen since 1969 The response of the strain to methyl benzoquate is illustrated in Fig. 3D and Table 2. The dose response relationship was similar to that found for the Weybridge, Beltsville and Elberfeld strains. Oocyst production was completely suppressed by 5 p.p.m. of methyl benzoquate. After 8 serial passages through birds treated with drug the strain had developed resistance. Restoration of sensitivity of the Houghton strain to methyl benzoquate Eleven of the 21 oocyst lines established in birds from an original inoculum of approximately 10 oocysts were completely suppressed by 40 p.p.m. methyl benzoquate. No oocyst production occurred and weight gains were 100% or more of non-infected non-medicated birds. Attempts were made to passage these lines by infecting birds given food medicated at 10 or 5 p.p.m. Oocyst production was completely suppressed. The remaining 10 oocyst lines were partially controlled by 40 p.p.m. methyl benzoquate with weight gains varying from 66 to 111 % of uninfected nonmedicated birds. Oocyst production was not suppressed in any of these lines. After a single passage in the presence of methyl benzoquate the drug failed to control infections in 9 of the lines. Weight gains varied from 14 to 57 % and were similar to infected, non-medicated birds. One oocyst line was still partially controlled by 40 p.p.m. methyl benzoquate. The failure of methyl benzoquate to control the infections after a single passage indicates these lines were contaminated with resistant organisms. Development of resistance by a sensitive culture of the Houghton strain established from 10 oocysts One of the 11 oocyst lines which had been completely controlled by 40 p.p.m. methyl benzoquate was selected for further study. Oocyst production was suppressed at concentrations of 5 p.p.m. and above (Table 2). The response to different concentrations of methyl benzoquate is illustrated in Fig. 3E. At concentrations of 5 p.p.m. and above, weight gain was similar to non-infected nonmedicated birds. After 8 serial passages through birds treated with drug the strain had developed resistance. DISCUSSION
The rate of development of resistance to the quinolones observed in this study was found to be similar to that published by other workers. When the Weybridge, Beltsville and Elberfeld strains of E. tenella were compared under similar conditions, the Weybridge and Elberfeld strains developed resistance to methyl benzoquate after six passages and the Beltsville after five. This difference of a single passage is not likely to be of significance. The cross resistance of the current 4
PAR
71
48
H. D. CHAPMAN
Houghton strain to other quinolones but not to robenidine and amprolium confirms the findings of other workers. The current Houghton strain was found to behave differently from the other strains studied, resistance appearing after a single passage in the presence of drug. Three possibilities may be put forward to explain this anomalous behaviour. First, there may be a true difference in the susceptibility of the strain to drugs, resistance after a single passage reflecting strain heterogenicity in E. tenella. Secondly, a low level of resistance may have been acquired by the strain due to accidental exposure to quinolones during its maintenance at Houghton Poultry Research Station. Finally, the strain may have become contaminated with oocysts already resistant despite rigorous techniques for isolation and maintenance. The fact that oocysts derived from a culture which had been frozen in liquid nitrogen since 1969 were fully sensitive to methyl benzoquate would appear to rule out the first possibility, and suggest that this partial resistance has been acquired since 1969. It is interesting that an embryo-adapted strain derived from the Houghton strain at this time also showed full sensitivity to methyl benzoquate and took 7 passages to develop resistance (Chapman, 1974). It is necessary to establish whether the partial resistance shown by the Houghton strain was due to a low level of resistance amongst all the oocysts of the strain, or due to the presence of a few totally resistant parasites in a sensitive population. This latter possibility was investigated by establishing infections with a few oocysts. If the infective dose is small enough then the resistant parasites should be diluted out. An oocyst line established in this manner should be fully sensitive to quinolones and take a number of passages to develop resistance. The results described in this paper suggest that this is indeed the case. Approximately 50 % of lines established from infections with 10 oocysts were fully sensitive and the one tested developed resistance after 8 serial passages. The Houghton strain frozen since 1969 also developed resistance after 8 serial passages. The greater number of passages taken to develop resistance may be related to the greater sensitivity to methyl benzoquate shown by both strains as judged by oocyst production (Table 2). The nine oocyst lines which developed resistance after a single passage evidently contained at least one resistant oocyst. On primary passage in treated chickens, the sensitive parasites in such an inoculum were all suppressed. Resistant parasites however, evidently multiplied and produced large numbers of oocysts resistant to methyl benzoquate. At the second passage only the resistant oocysts were present and consequently the drug failed to control the infection. It is not possible to establish the numbers of oocysts infecting the birds in these experiments and therefore the frequency of resistant parasites in the strain cannot be calculated. Accidental contamination of Houghton Poultry Research Station chicken food with quinolone anticoccidials has occurred on several occasions in the past. It is possible that the resistant parasites in the Houghton strain have arisen as a result of this contamination. For this reason, a special diet has been adopted (see Methods). Alternatively, resistant parasites may have contaminated stock cultures from outside despite rigorous precautions to maintain their isolation.
Resistance of E. tenella to quinolone drugs
49
Foure & Bennejean (1973) have reported similar rapid development of resistance to quinolones. It is probable that a similar explanation is applicable in this case. It would like to thank Mr P. Townsend and Mrs B. Hill for their technical assistance in this study. REFERENCES CHAPMAN, H. D.
(1974). Use of chick embryo infections for the development of drug resistance in Eimeria tenella. Parasitology (in the Press). FOUBE, N. & BENNEJEAN, G. (1973). Les chimioresistances acquises vis a vis des derives des quinoleines et de la pyridine. Symposium international aur les coccidioses, Tours 1973. JEFFEBS, T. K. (1974). Eimeria tenella: Incidence, distribution and anti-coccidial drug resisance of isolants in major broiler producing areas. Avian Diseases 18, 74-84. JOYNEE, L. P. (1970). Coccidiosis: problems arising from the development of anticoccidial drug resistance. Experimental Parasitology 28, 122-8. JOYNEB, L. P. & NOBTON, C. C. (1973). The immunity arising from continuous low level infection with Eimeria tenella. Parasitology 67, 333-40. LONG, P. L. (1968). The pathogenic effects of Eimeria praecox and E. acervulina in the chicken. Parasitology 58, 691-700. LONG, P. L. (1972). Eimeria tenella: Reproduction, Pathogenicity and immunogenicity of a strain maintained in chick embryos by serial passage. Journal of Comparative Pathology 82, 429-37. LONG, P. L. & ROWELL. J. G. (1958). Counting oocysts of chicken coccidia. Laboratory Practice 7, 515-9. MCLOUGHLIN, D. K. (1970). Efficacy of buquinolate against ten strains of Eimeria tenella and the development of a resistant strain. Avian Diseases 14, 126-30. MCLOUGHLIN, D. K. & CHUTE, M. B. (1971). Efficacy of decoquinate against eleven strains of Eimeria tenella and development of a decoquinate resistant strain. Avian Diseases 15, 342-5. MCLOUGHLIN, D. K. & CHUTE, M. B. (1973). Efficacy of nequinate against thirteen strains of E. tenella and the development of a nequinate resistant strain. Avian Diseases 17, 717-21. MCLOUGHLIN, D. K. & GABDINEB, J. L. (1961). Drug resistance in Eimeria tenella. 1. The experimental development of a glycarbylamide resistant strain. Journal of Parasitology 47, 1001-6. MCMANUS, E. C, CAMPBELL, W. C. & CUCKLEB, A. C. (1968). Development of resistance to quinolone coccidiostats under field and laboratory conditions. Journal of Parasitology 54 1190-3. MILLABD, B. J. (1970). Observations on a drug resistant strain of Eimeria tenella. Research in Veterinary Science 11, 394-7. RYLEY, J. F. & BBTTS, M. J. (1973). Chemotherapy of chicken coccidiosis. In Advances in Pharmacology and Chemotherapy, vol. xi (ed. S. Garattini, A. Goldin, F. Hawking and I. J. Kopin), pp. 221-93. New York: London, Academic Press.
Printed in Great Britain 4-2
41
Eimeria tenella in chickens: development of resistance to quinolone anticoccidial drugs H. D. CHAPMAN Houghton Poultry Research Station, Houghton, Huntingdon, Cambs. (Received 4 December 1974) SUMMARY
The development of drug resistance by the present Houghton strain of Eimeria tenella to the quinolones, methyl benzoquate and buquinolate, was found to take place after a single experimental passage. The development of resistance was independent of drug selection pressure and showed cross resistance to other quinolones, but not to amprolium and robenidine. When the Weybridge, Beltsville and Elberfeld strains of E. tenella were compared under similar laboratory conditions, the Weybridge and Elberfeld strains developed resistance to methyl benzoquate after 6 passages and the Beltsville after 5. Studies on the response of the Houghton strain to methyl benzoquate and buquinolate revealed that the drugs did not completely control the infection as measured by weight gain and that oocyst production was not suppressed. These observations indicate that the strain had already acquired some resistance to these drugs. This was confirmed by examining the resistance to methyl benzoquate of a culture of the Houghton strain of E. tenella which had been stored frozen in liquid nitrogen since 1969. This showed full sensitivity to the drug and developed resistance after 8 passages. This suggests that drug tolerance has been acquired by the Houghton strain since 1969. Oocyst lines were established from the Houghton strain by infecting single birds with approximately 10 oocysts. Eleven of these lines were found to be sensitive to methyl benzoquate, and nine to give rise to resistant parasites. It is concluded that the Houghton strain is contaminated by a small number of resistant oocysts which can be eliminated from a culture by dilution of the challenge inoculum. One of these Houghton oocyst lines, sensitive to methyl benzoquate, developed resistance after 8 serial passages. INTRODUCTION
Several workers have shown the apparent ease with which the coccidia acquire resistance to the quinolones; resistance of Eimeria to buquinolate, decoquinate and methyl benzoquate developing after 4-6 passages in the presence of drug (McLoughlin, 1970; McLoughlin & Chute, 1971, 1973). Field strains of E. tenella resistant to quinolones have been described (Millard, 1970; McManus, Campbell & Cuckler, 1968) and in a recent study of the incidence of drug resistance in the field, Jeffers (1974) found that resistance to decoquinate and buquinolate was present in 40-60% of E. tenella isolates examined. McManus et al. (1968) found that species of Eimeria differ in their rate of development of resistance. In studies
42
H. D. CHAPMAN
with amquinolate resistance developed after 4 passages in E. tenella, E. brunetti and E. maxima and after 8 passages in E. acervulina but no information was given on possible differences between strains of the same species. According to Ryley & Betts (1973), different strains of one species isolated from different localities usually vary in their sensitivity to a drug with which they have not had prior contact. Joyner (1970) has described differences in drug efficacy against the Houghton and Weybridge strains of E. tenella and has suggested that inherent heterogenicity among field strains may influence the development of drug resistance. In this paper, the development of resistance by the Houghton strain of E. tenella to the quinolones, methyl benzoquate and buquinolate, is described. A comparison is made between the Weybridge, Beltsville and Elberfeld strains of this species. The anomalous behaviour of the Houghton strain is discussed and the restoration of sensitivity of this strain to methyl benzoquate is described. METHODS
Animals. Twelve-day-old Light Sussex chickens were used. They were kept in wire floored metal cages and maintained as described by Long (1968). Drugs were incorporated into a standard ration supplemented with vitamin K. The ration was formulated for experimental work with coccidia; details of its composition have been given by Ryley & Betts (1973). The food was prepared in a special mill used only for experimental diets making contamination with anticoccidial drugs extremely unlikely. Infective material. The Houghton strain of E. tenella chosen for study has been maintained in this laboratory by serial passage through chickens. The Elberfeld strain was supplied by Dr A. Haberkorn and a culture of the Houghton strain frozen in liquid nitrogen since 1969 by Dr L. P. Joyner. Graded infective doses of oocysts were achieved by serial dilution of a freshly prepared inoculum which had been accurately counted. All suspensions of oocysts were stirred whilst inoculating the birds to ensure that each received a similar dose. After 7 days the birds were killed, weighed and oocysts were harvested from the caeca using the method described by Long (1972). Oocysts in the faeces of infected groups of birds were counted by methods described by Long & Rowell (1958). Experimental procedure for developing drug resistance. Birds were allocated to groups of ten using a restricted randomization procedure which approximately equalized average initial weights, and inoculated with 100000 oocysts. The number of passages required to develop resistance was then established by serially passaging the coccidia in the presence of sub-optimal concentrations of drug. Birds were infected with 100000 oocysts at each passage. The design of the experiments was based upon procedures established by McLoughlin & Gardiner (1961). The essential feature was the division of the original strain into two oocyst lines, one passaged in the presence of the drug to which resistance is to be developed, the other passaged for the same number of times in the absence of drug. This latter line served as a reference standard,
Resistance of E. tenella to quinolone drugs
43
against which the response of medicated chicks to infection could be measured, and served to ensure ttnat no change in VnruYence accompanied. \3&fi> seqvxence oi passages. The response of this line to drugs after resistance had been developed in the 'resistant' line also enabled a check to be made that the strain had retained its original sensitivity despite the sequence of passages. Both weight gain and oocyst production were used as parameters for measuring drug activity. Weight gains were expressed as a percentage of the weight gain of uninfected, non-medicated birds. The % weight gain of birds infected and non-medicated is also included in the figures. Restoration of sensitivity of the Houghton strain to methyl benzoquate. Twentyfour birds were each given 10 oocysts. The dose of 10 oocysts was only approximate, since attempts to verify the numbers present by direct counting after the method of Joyner & Norton (1973) were unsuccessful. Twenty-one of these chickens became infected and small numbers of oocysts were harvested. Each oocyst line established in this way was then re-passaged in 2 birds to build up the number of oocysts and handled separately throughout the experiment. Strict precautions were taken to ensure that cross contamination did not occur. The response of each line to 40 parts per million (ppm) methyl benzoquate was then established by inoculating groups of 5 birds individually with 100000 oocysts. In some of these lines oocyst production was completely suppressed. Further attempts were made to passage them at lower drug concentrations. In those lines where oocysts were recovered from the primary passage, the degree of resistance was evaluated by passage for a second time in the presence of drug.
RESULTS
Development of resistance by the Houghton strain to methyl benzoquate and buquinolate The response of the Houghton strain to methyl benzoquate is illustrated in Fig. 1 A. Primary passage in the presence of different concentrations of this drug resulted in a dose-response relationship in which maximum activity was found at concentrations of 1 p.p.m. and above. Below this concentration drug activity declined to a level similar to infected non-medicated birds. A similar relationship was found for buquinolate with maximum activity at 32 p.p.m. and above and little activity below 10 p.p.m. (Fig. IB). Oocysts were harvested from birds fed 40 p.p.m. methyl benzoquate and 82.5 p.p.m. buquinolate and the dose-response relationship again established (Fig. 1A, B). All concentrations of drug used failed to control infections and weight gains of medicated groups were similar to infected non-medicated birds. Development of resistance by the Houghton strain passaged at different concentrations of drug The strain was passaged in birds given drug in the food at concentrations ranging from 0-1 to 40 p.p.m. methyl benzoquate and 4-0 to 500 p.p.m. buquinolate. After a single passage, oocysts were harvested from the various lines and the degree of resistance established by measuring the response to 40 p.p.m. methyl
44 1 VU —
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f\
80
Weight
60
U
/
1
40
\
20
10
0-1
100 1
100
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1000
Drug concentration
Fig. 1. Response of the Houghton strain of Eimeria tenella to drugs. (A) Methyl benzoquate. (B) Buquinolate. Key: % weight gain of infected non -medicated birds included in brackets after the passage number. 9 — 0 , primary passage (25%); O—O> 2nd passage (32%). Drug concentrations plotted on a log scale. 100 i -
80
I
60
DO
40
20
I
0-1
I
I
10 10 100 Drug concentration used in passage
1000
Fig. 2. Response of the Houghton strain passaged at different drug concentrations to 40 parts per million (ppm) methyl benzoquate and 82-5 ppm buquinolate. 0 — 0 , methyl benzoquate; O—O> buquinolate. Weight gain of infected, nonmedicated birds was 32 % of non-infected controls. benzoquate and 82-5 p.p.m. buquinolate. Results are illustrated in Fig. 2. In those lines where the drug had initially controlled the infections weight gains were similar to infected non-medicated birds indicating the development of resistance. In those lines where no control of the infection had been found (0-1 p.p.m. methyl benzoquate, 4-0-8-0 p.p.m. buquinolate) resistance did not develop.
Resistance of E. tenella to quinolone drugs
45
Table 1. Effect of drugs on two resistant lines of the Houghton strain of Eimeria tenella and a control line which had been passaged for the same number of times in the absence of drug Weight gain-% uninfected non-medicated birds Drug concentration (ppm) Methyl benzoquate 40 Buquinolate 82-5 Decoquinate 40 Robenidine 33 Amprolium 125 Non-medicated
Methyl benzoquate resistant
Buquinolate resistant
Control
16 35 20 103 93 39
24 42 38 96 98 32
84 81 95 102 91 25
Cross resistance The methyl benzoquate and buquinolate resistant lines were fully cross resistant to each other and to decoquinate but showed no cross resistance to robenidine and amprolium (Table 1). Development of resistance by the Weybridge, Beltsville and Elberfeld strains of E. tenella The response to methyl benzoquate is illustrated in Fig. 3A-C. At concentrations of 10 p.p.m. and above, methyl benzoquate showed greater activity against these strains than had been found for the Houghton strain. Weight gains were approximately 100 % that of uninfected non-medicated birds. Both Beltsville and Elberfeld strains showed weight gains of approximately 80 % at 1 p.p.m. with similar activity at 5 p.p.m. for the Weybridge strain. Declining activity was found at lower drug concentrations. Oocyst production was initially completely suppressed by 10 p.p.m. methyl benzoquate and so the strains were passaged at the maximum concentration of drug which would permit sufficient oocysts to be harvested to continue the passage. After 6 serial passages with the Weybridge and Elberfeld strains and 5 passages with the Beltsville strain, weight gains declined to levels similar to infected non-medicated birds. The dose response relationship was then established at different drug concentrations (Fig. 3A-C). The response of the Elberfeld strain (Fig. 3C) to different concentrations of drug showed more variation than the other strains. In all three strains, complete failure to control infections was found at all drug levels indicating the development of resistance. Oocyst production by the various strains of E. tenella at different concentrations of methyl benzoquate Control of the Houghton strain by methyl benzoquate has been shown to be incomplete as judged by the weight gain of infected medicated birds. This was further studied by examining oocyst production of the strains of E. tenella in birds given different concentrations of drug (Table 2). Oocyst production by the Weybridge, Beltsville and Elberfeld strains was completely suppressed by 10 p.p.m.
46
H. D. CHAPMAN A
100
^
80
E
60
rc
DO
l>40 *
20
0-1
10
10
100 0-1
10 10 Drug concentration
100 0 1
10
10
100
- E 80 cN c
/ 60 -
/
So JZ
1
40
/
rf
^-^°
20 -
01
1
1
10
10
1
10 100 0-1 Drug concentration
10
100
Fig. 3. Response of different strains of Eimeria tenella to methyl benzoquate. (A) Weybridge: 0 — # , primary passage (32%); O—O, 6th passage (24%). (B) Beltsville: # — • , primary passage (21%); O—O, 5th passage (39%). (C) Elberfeld: • — • , primary passage (28%); O—O, 6th passage (23%). (D) Houghton (1969): # — • , primary passage (37%); O—O. 8th passage (31 %). (E) Houghton (sensitive isolate): # — # , primary passage (40%); O—O, 8th passage (22%). Table 2. Oocyst production of different strains of Eimeria tenella in response to different concentrations of methyl benzoquate Oocysts produced (millions/bird) Drug concentration (ppm in food) Strain of E. tenella Houghton (current) Weybridge Beltsville Elberfeld Houghton (1969) Houghton (sensitive isolate)
40
20
10
5
1
0
23-6
17-9
15-7
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
24- 2 16- 1 1- 0 1- 0 0 0
12-1 29-0 10-2 10-2 9-8 39-6
28-8 17-4 24-0 26-6 11-9 29-8
Resistance of E. tenella to quinolone drugs
47
methyl benzoquate, but no control of the Houghton strain was obtained. These results indicated that some resistance had already been acquired by this strain. Development of resistance by the Houghton strain prepared from material frozen since 1969 The response of the strain to methyl benzoquate is illustrated in Fig. 3D and Table 2. The dose response relationship was similar to that found for the Weybridge, Beltsville and Elberfeld strains. Oocyst production was completely suppressed by 5 p.p.m. of methyl benzoquate. After 8 serial passages through birds treated with drug the strain had developed resistance. Restoration of sensitivity of the Houghton strain to methyl benzoquate Eleven of the 21 oocyst lines established in birds from an original inoculum of approximately 10 oocysts were completely suppressed by 40 p.p.m. methyl benzoquate. No oocyst production occurred and weight gains were 100% or more of non-infected non-medicated birds. Attempts were made to passage these lines by infecting birds given food medicated at 10 or 5 p.p.m. Oocyst production was completely suppressed. The remaining 10 oocyst lines were partially controlled by 40 p.p.m. methyl benzoquate with weight gains varying from 66 to 111 % of uninfected nonmedicated birds. Oocyst production was not suppressed in any of these lines. After a single passage in the presence of methyl benzoquate the drug failed to control infections in 9 of the lines. Weight gains varied from 14 to 57 % and were similar to infected, non-medicated birds. One oocyst line was still partially controlled by 40 p.p.m. methyl benzoquate. The failure of methyl benzoquate to control the infections after a single passage indicates these lines were contaminated with resistant organisms. Development of resistance by a sensitive culture of the Houghton strain established from 10 oocysts One of the 11 oocyst lines which had been completely controlled by 40 p.p.m. methyl benzoquate was selected for further study. Oocyst production was suppressed at concentrations of 5 p.p.m. and above (Table 2). The response to different concentrations of methyl benzoquate is illustrated in Fig. 3E. At concentrations of 5 p.p.m. and above, weight gain was similar to non-infected nonmedicated birds. After 8 serial passages through birds treated with drug the strain had developed resistance. DISCUSSION
The rate of development of resistance to the quinolones observed in this study was found to be similar to that published by other workers. When the Weybridge, Beltsville and Elberfeld strains of E. tenella were compared under similar conditions, the Weybridge and Elberfeld strains developed resistance to methyl benzoquate after six passages and the Beltsville after five. This difference of a single passage is not likely to be of significance. The cross resistance of the current 4
PAR
71
48
H. D. CHAPMAN
Houghton strain to other quinolones but not to robenidine and amprolium confirms the findings of other workers. The current Houghton strain was found to behave differently from the other strains studied, resistance appearing after a single passage in the presence of drug. Three possibilities may be put forward to explain this anomalous behaviour. First, there may be a true difference in the susceptibility of the strain to drugs, resistance after a single passage reflecting strain heterogenicity in E. tenella. Secondly, a low level of resistance may have been acquired by the strain due to accidental exposure to quinolones during its maintenance at Houghton Poultry Research Station. Finally, the strain may have become contaminated with oocysts already resistant despite rigorous techniques for isolation and maintenance. The fact that oocysts derived from a culture which had been frozen in liquid nitrogen since 1969 were fully sensitive to methyl benzoquate would appear to rule out the first possibility, and suggest that this partial resistance has been acquired since 1969. It is interesting that an embryo-adapted strain derived from the Houghton strain at this time also showed full sensitivity to methyl benzoquate and took 7 passages to develop resistance (Chapman, 1974). It is necessary to establish whether the partial resistance shown by the Houghton strain was due to a low level of resistance amongst all the oocysts of the strain, or due to the presence of a few totally resistant parasites in a sensitive population. This latter possibility was investigated by establishing infections with a few oocysts. If the infective dose is small enough then the resistant parasites should be diluted out. An oocyst line established in this manner should be fully sensitive to quinolones and take a number of passages to develop resistance. The results described in this paper suggest that this is indeed the case. Approximately 50 % of lines established from infections with 10 oocysts were fully sensitive and the one tested developed resistance after 8 serial passages. The Houghton strain frozen since 1969 also developed resistance after 8 serial passages. The greater number of passages taken to develop resistance may be related to the greater sensitivity to methyl benzoquate shown by both strains as judged by oocyst production (Table 2). The nine oocyst lines which developed resistance after a single passage evidently contained at least one resistant oocyst. On primary passage in treated chickens, the sensitive parasites in such an inoculum were all suppressed. Resistant parasites however, evidently multiplied and produced large numbers of oocysts resistant to methyl benzoquate. At the second passage only the resistant oocysts were present and consequently the drug failed to control the infection. It is not possible to establish the numbers of oocysts infecting the birds in these experiments and therefore the frequency of resistant parasites in the strain cannot be calculated. Accidental contamination of Houghton Poultry Research Station chicken food with quinolone anticoccidials has occurred on several occasions in the past. It is possible that the resistant parasites in the Houghton strain have arisen as a result of this contamination. For this reason, a special diet has been adopted (see Methods). Alternatively, resistant parasites may have contaminated stock cultures from outside despite rigorous precautions to maintain their isolation.
Resistance of E. tenella to quinolone drugs
49
Foure & Bennejean (1973) have reported similar rapid development of resistance to quinolones. It is probable that a similar explanation is applicable in this case. It would like to thank Mr P. Townsend and Mrs B. Hill for their technical assistance in this study. REFERENCES CHAPMAN, H. D.
(1974). Use of chick embryo infections for the development of drug resistance in Eimeria tenella. Parasitology (in the Press). FOUBE, N. & BENNEJEAN, G. (1973). Les chimioresistances acquises vis a vis des derives des quinoleines et de la pyridine. Symposium international aur les coccidioses, Tours 1973. JEFFEBS, T. K. (1974). Eimeria tenella: Incidence, distribution and anti-coccidial drug resisance of isolants in major broiler producing areas. Avian Diseases 18, 74-84. JOYNEE, L. P. (1970). Coccidiosis: problems arising from the development of anticoccidial drug resistance. Experimental Parasitology 28, 122-8. JOYNEB, L. P. & NOBTON, C. C. (1973). The immunity arising from continuous low level infection with Eimeria tenella. Parasitology 67, 333-40. LONG, P. L. (1968). The pathogenic effects of Eimeria praecox and E. acervulina in the chicken. Parasitology 58, 691-700. LONG, P. L. (1972). Eimeria tenella: Reproduction, Pathogenicity and immunogenicity of a strain maintained in chick embryos by serial passage. Journal of Comparative Pathology 82, 429-37. LONG, P. L. & ROWELL. J. G. (1958). Counting oocysts of chicken coccidia. Laboratory Practice 7, 515-9. MCLOUGHLIN, D. K. (1970). Efficacy of buquinolate against ten strains of Eimeria tenella and the development of a resistant strain. Avian Diseases 14, 126-30. MCLOUGHLIN, D. K. & CHUTE, M. B. (1971). Efficacy of decoquinate against eleven strains of Eimeria tenella and development of a decoquinate resistant strain. Avian Diseases 15, 342-5. MCLOUGHLIN, D. K. & CHUTE, M. B. (1973). Efficacy of nequinate against thirteen strains of E. tenella and the development of a nequinate resistant strain. Avian Diseases 17, 717-21. MCLOUGHLIN, D. K. & GABDINEB, J. L. (1961). Drug resistance in Eimeria tenella. 1. The experimental development of a glycarbylamide resistant strain. Journal of Parasitology 47, 1001-6. MCMANUS, E. C, CAMPBELL, W. C. & CUCKLEB, A. C. (1968). Development of resistance to quinolone coccidiostats under field and laboratory conditions. Journal of Parasitology 54 1190-3. MILLABD, B. J. (1970). Observations on a drug resistant strain of Eimeria tenella. Research in Veterinary Science 11, 394-7. RYLEY, J. F. & BBTTS, M. J. (1973). Chemotherapy of chicken coccidiosis. In Advances in Pharmacology and Chemotherapy, vol. xi (ed. S. Garattini, A. Goldin, F. Hawking and I. J. Kopin), pp. 221-93. New York: London, Academic Press.
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