Resistance to anticoccidial drugs in fowl

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(1988) J. Parasitol. 74, 614 617 24 Giordano, D.J., Tritschler, J.P. and Coles, G.C. (1988) Vet. Parasitol. 30, 139 148 25 Shoop, W.L et al. (1990) J. Pdrasitol. 76, 186-189 26 Taylor, M.A. (1990) Res. Vet. Sci.49, 198 202 27 Lacy, E. et al. (1990) in Resistance of Parasites to Antiparasitic Drugs (Boray, J.C., Martin, P.J. and Roush, R.T., ads), pp 177 184, MSD AGVET Merck & Co. 28 Hubert, J. and Kerboeuf, D. (1992) Vet. Rec. 130, 442 446 29 Gill, G.H. et al. (1991) Int. J. Parasitol. 21, 771 776 30 Echevarria, F.A.M., Gennari, S.M. and Tait, A. (1992) Vet. ParasitoL44, 87 95 31 Scott, E.W. and Armour, J. (1991) Vet. Rec. 128, 346 349

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32 DeVaney, J.A., Craig, T.M. and Rowe, L.D. (I 992) Int.J. Parasitol. 22, 369-376 33 Pankavich, J.A., Berger, H. and Simkins, K.L. (1992) Vet. Rec. 130,241-243 34 Craig, T.M. et el. (1992) Vet. Parasitot. 41, 329 333 35 Shoop, W.L (1992) Vet. Rec. 130,563 36 Shoop,W.L (1992) Vet. Rec 131,375 37 Shoop,W.L et al. Vet. Rec. (in press) 38 Conder, C.A., Thompson, D.P. and Johnson, S.S.Vet. Rec. (in press) 39 Duce, I.R. and Scott, R.H. (1985) Br. j Phan macol. 85, 395 -401 40 Martin, R.J.and Pennington, A.J. (1989) Br. j Pharmacol. 98, 747-756 41 Egerton, JR. et al. (1980) Br Vet. J. 136, 88 97 42 Blair, LS., Malatesta, P.F. and Ewanciw, E).V.

(1983) Am.j. Vet. Res.44, 475 477 43 Klei, T.R., Torbert, B.J.and Ochoa, R. (1980) J. Parasitol. 66, 859 861 44 Egerton, J.R. et al. (1981) Vet. ParasitoL 8, 83 88 45 Forsyth, K.P., Mitchell, G.F. and Copeman, D.B. (1984) Exp. Parasitol. 58, 41 55 46 Coles, G.C. and Roush, RT. (1992) Vet. Rec. 130, 505 510 47 van Wyk, J.A. and van Schalkwyk, P.C. (1990) Vet. Parasitot. 35, 61 69 48 Prichard, R.K. et al. (t980) Aust. Vet. J. 56, 239 251 Wesley L.. Shoop is at Merck Research Laboratories, PO Box 2000, Merck and Co., Rahway, NJ 07065, USA.

Resistance to Anticoccidial Drugs in Fowl H.D. Chapman Resistance has been encountered wherever drugs have been used extensively for the control of parasitic infections. The poultry industry is dependent upon drugs for the control of coccidiosis, a major dis ease of chickens caused by protozoan parasites of the genus Eimeria. In modem poultry production, drugs are used prophylactically for the prevention of coccidiosis by including them in the diet. This has inevitably led to the development of resistance. We have been fortunate in that new drugs have become available to replace those to which resistance has developed, but this situation is unlikely to continue. The problem of drug resistance, discussed here by David Chapman, has provided impetus for the development of new approaches (such as vaccination) for the control of coccidiosis.

Despite the extensive use of drugs, it has proved impossible to eradicate coccidiosis. Medication via the feed has proved to be convenient, labour-saving and cost-effective, and has been a major factor in enabling large numbers of chickens to be reared in the intensive conditions of the modem poultry industry. Horton-Smith was the first to suggest that resistance might develop to anticoccidial drugs (C. Horton-Smith, abstract*). However, Joyner et al. ~ stated in 1963 that resistance still does not represent any serious problem in the control of coccidiosis in the field. *9th World's Poultry Congress (1951) Paris, France © 1993,ElsevierSciencePubhshersLtd, (UK)

Today, this is no longer the case. Many drugs have been introduced, but resistance has arisen to all of them.

Selection of Resistance The life cycle of Eimeria involves a period of asexual multiplication (schizogony) in the epithelial cells of the intestine of the host, followed by a sexual phase (gametogony), resulting in the production of oocysts that are excreted in the feces. Most drugs inhibit asexual stages in the cycle, such as the first- or second-generation schizont. Complexities involved in the selection of resistant forms in diploid organisms, such as the degree of dominance of resistance genes, are absent in Eimeria because the asexual stages are haploid. Resistant mutants will, therefore, be immediately selected in the presence of a drug at the expense of sensitive forms. The coccidia have an enormous capacity to multiply in the intestine (one oocyst of E. tenella can give rise to more than 100 000 oocysts in a single generation), and so resistance can rapidly become the dominant phenotype. Ionophorous antibiotics (monensin, salinomycin, narasin, lasalocid and maduramicin) are currently the most widely used drugs for the control of coccidiosis. It has proved difficult to induce resistance to these drugs in the laboratory 2, and resistance to them was slow to develop in the field. A new compound, diclazuril, has recently been introduced for the con-

trol of coccidiosis. The experimental development of resistance to diclazuril, in E. tenella, was compared with that to methyl benzoquate and amprolium, drugs to which resistance develops rapidly and slowly, respectively 3. Complete resistance developed to methyl benzoquate after six passages, but only partial resistance developed to amprolium and diclazuril after ten passages, It will be interesting to see how rapidly resistance develops to diclazuril in the field. Three species of Eimeria (E. acervulina, E. tenelta and E. maxima) are commonly encountered, and are capable of causing economic loss. In the case of diclazuril, resistance developed in E. acervulina and E. tenella, but not in E. maxima, suggesting that resistance to diclazuril is more likely to develop in E. acervulina and E. tenella than in E. maxima. Although it may be possible to obtain an approximate indication of the likelihood of resistance developing to a new drug, the selection pressures to which parasites are exposed in the laboratory will be very different from those in the field. It is not possible, therefore, to extrapolate directly from the former to the latter. The population dynamics of Eimeria species deserve study, since we know little about the factors involved in the spread of resistance.

Mechanisms of Resistance Although information is available on the mode of action and biochemical

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pathways inhibited by some anticoccidial drugs, explanations for the mechanisms of resistance are either speculative or unavailable. Quinolones, such as methyl benzoquate, block electron transport in the parasite mitochondrion. Resistance develops readily to these drugs, but the mechanism involved is unknown 4. Amprolium inhibits the transport of thiamine across the parasite cell membrane s, The mechanism of resistance possibly involves modification of a target receptor so that its sensitivity to inhibition is decreased. A similar mechanism of resistance has been proposed for pyrimethamine (component of potentiated sulphonamides) that inhibits dihydrofolate reductase in the folic acid pathway 6. In other protozoa, progress has recently been made in understanding the mechanism of resistance to pyrimethamine at the molecular level7. Hopefully, one day it will be possible to identify and characterize the genes responsible for resistance to anticoccidial agents. Ionophores are capable of transporting cations through biological membranes and affect a diverse range of cellular processes dependent upon ion transport. These drugs are accumulated by sporozoites before they penetrate host cells, but destruction of the parasite may occur before or after host cell penetration 8. It has been shown that the uptake of monensin by sporozoites resistant to the drug was significantly less than that of sensitive sporozoites 9. The amount of drug required to inhibit development of resistant forms was 20

/

•..e

to 40 times higher than for sensitive parasites. Differences in ionophore accumulation may, therefore, be responsible for the expression of resistance, but the mechanisms involved are not understood. It is likely that fundamental changes in the biophysical properties of the cell wall of the parasite would be required to cope with a nonspeciflc action upon trans-membrane cation transport. This might not readily be accomplished even with intense selection pressure for resistance. Ideally, knowledge of these mechanisms should help in the development of strategies for countering resistance, but there are few examples, so far, in which such information has led to improved control of parasitic infections. Cross-resistance

Compounds that share a similar mode of action may also share resistance (cross-resistance). Ionophores are believed to have the same mode of action and most workers consider that cross-resistance occurs between these compounds. The effect of salinomycin, narasin, monensin and lasalocid upon oocyst production of a line of E. tenella to which resistance to monensin had been developed, is shown in Fig. I. Although fewer oocysts were produced by birds given lasalocid, substantial numbers were found with all four drugs, suggesting that resistance is shared. Other studies suggest that lasaIocid is able to control strains that are resistant to monensin, and it has been

~.~°\e

:.e

10 8 cO 0

o Q_

107

>, 0 0

o

106 Ionophores Fig. I. The effect of various ionophores upon oocyst production after inoculation of chickens with a line ofEimeria tenella that is resistant to monensin.

proposed that the mode of action of lasalocid, which is capable of transport ing divalent as well as monovalent ions, may be somewhat different from that of monensin I°. Cross-resistance has been reported between maduramicin and other ionophores but some believe that this drug is effective against strains resistant to these drugs ~. Clearly, further work is required on cross-resistance to the various ionophores. It is important that a new drug is able to control strains resistant to existing compounds. Diclazuril, for example, is effective against strains that are resistant to eight other drugs 3. Diclazuril may share resistance with toltrazuril, however, since both compounds have a similar chemical structure and may share a common mode of action. Multiple Resistance

Cross-resistance should be distinguished from multiple resistance, in which coccidia may be resistant to more than one drug, even though they have different modes of action. The occurrence of multiple resistance was investigated in 15 isolates of E. tenella from broiler farms in the UK (Table I). Eight drugs with different modes of action were tested. Five of the isolates were resistant or partially resistant to four drugs, and two isolates were resistant or partially resistant to five drugs. Multiple resistance probably arises as a result of sequential exposure to the compounds in question in successive flocks. Multiple resistance may also occur as a result of genetic recombination between lines of parasites that are resistant to different drugs. There has been one attempt to produce a recombinant that is resistant to more than two drugs ~2. A line of E. tenella resistant to robenidine, decoquinate and amprolium was produced, but a line that was resistant to five drugs could not be obtained. An unsuccessful attempt has been made to obtain, by genetic recombination, a line of E. max ima that is resistant to both methyl benzoquate and clopidol ~3. Although cross-resistance does not occur between these compounds, they may act against related phases of the metabolism of Eimena, and it is possible that genes determining resistance to them are closely linked on the same chromosome. In this case it would be difficult to obtain resistance to both

Parasitology Today, vol 9, no. 5, 1993

drugs. Despite the failure to obtain a line of E. maxima resistant to methyl benzoquate and clopidol by genetic recombination, strains of this species that are resistant to both compounds do occur in the field. In this case, resistance probably results from successive exposure to each drug. Stability of Resistance

Most studies have shown that resistance is stable, but resistance may be lost if a drug is no longer used; possibly as a result of the outgrowth of resistant parasites by sensitive forms. For example, when equal numbers of oocysts of a drug-sensitive and drug-resistant strain of E. tenella were introduced into a pen of unmedicated chickens, the drug-sensitive strain subsequently became the predominant proportion of the population ~4. We do not know if this phenomenon has occurred in the field. In the UK, several older drugs (including robenidine and methyl benzoquate) were found to be effective against recent field isolates of Eimeria p5 even though their use had been discontinued, primarily because of drug resistance (Table I). Thus, restoration of sensitivity to some compounds may occur. Nevertheless, such drugs should be used sparingly because resistance would be likely to emerge rapidly if they were re-introduced. Reversal of resistance may occur if strains of Eimeria resistant to certain drugs are passaged in chickens that are given another compound. An example is the loss of resistance to decoquinate following the use of clopido116. It is not known if reversal of resistance occurs in practice. C o m b i n a t i o n s of D r u g s

It is thought that resistance is less likely to develop when two or more compounds are used in combination rather than individually. Combinations of as many as four drugs have been employed for the control of coccidiosis, but there is little evidence that the development of resistance has been delayed. In most cases the components of drug mixtures have already been used independently, and consequently, resistance to them has developed. A combination of 50 ppm nicarbazin and 50ppm narasin (Maxiban) has recently been introduced. It will be interesting to see how long this combination of drugs remains effective,

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Table I. Incidence of resistance in field isolates of Eimeria tenella to eight anticoccidial drugs

Drug

Isolate a I 2 3 4 5 6 7 8 9

Diclazuril Robenidine Methyl benzoquate Nicarbazin Clopidol Amprolium Dinitolmide Narasin No. of drugs to which resistant/ partially resistant

No. of isolates resistant/ partially resistant 101112131415

S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S P S S S S R R

S S S R R

S S S S S S S S P R R R R R R R R R R R R

S S R R R

S S R R R

P S R R R

P P S S S S R R R R P R R R R R R R R R R

S P R R

P R R R

4 4

13 15 15

2 2 3 3 3 3 3 3 4 4 4 4 4 5 5

aAbbreviations: R, resistant; P, partially resistant; S, sensitive.

because nicarbazin is included in the mixture at a concentration lower than that normally used for the control of coccidiosis and strains resistant to narasin are already present in the field (Table I). A l t e r n a t i o n of D r u g s and Vaccines

employed prophylactically for the prevention of the disease (eg. in rabbits). So far, there have been few reports of drug resistance in mammalian and avian hosts other than poultry. Drug resistance is likely to prove a problem wherever intensive systems of production are employed. Conclusion

Two or more drugs are often used within a single crop, and alternation of drugs from one crop of birds to another (rotation) has been widely practiced. It is not known if this has resulted in a delay in the development of resistance to the compounds employed. Oocysts of resistant parasites may survive in the litter for the life time of a single crop and, therefore, short periods of alternation between drugs are unlikely to result in a delay in the development of resistance. Vaccines based upon live oocysts of virulent or 'attenuated' parasites are available in various countries for the control of coccidiosis w. Alternation of the use of drugs with such vaccines might delay the development of resistance, and allow the replacement of resistant strains with drug-sensitive vaccine strains. It remains to be seen if this is a practical approach to the problem of drug resistance. O t h e r Hosts

Drugs are used therapeutically for the treatment of clinical coccidiosis in domestic livestock (eg. cattle and sheep), and in some cases are

Resistance is recognized as a major limitation of the successful use of drugs for the control of coccidiosis. As resistance has developed to established compounds, new ones have been discovered, but how long this can continue is of concern. Vaccines, whether based on live parasites or recombinant DNA technology, may offer the best alternative to the use of drugs. The integration of chemotherapeutic and immunologic methods of control should be considered. A better understanding of the epizootology of Eimena, the mode of action of drugs and mechanisms of resistance to them, and the mechanisms of immunity, should lead to improved methods for the control of coccidiosis. Acknowledgement This paper was published with the approval of the Director of the Agricultural Experiment Station.

References I Joyner, L.P., Davies, S.F.M. and Kendall, S.B. (1963) in Expenrnental Chemotherapy Vol. I (Schnitzer, R.J. and Hawking, F., eds), pp 445486, Academic Press 2 Chapman, HD. (1984) Parasitology 89, 9 16 3 Chapman, H.D. (1989) Parasitology 99, 189-t92

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4 Wang, C.C. (1975) Biochim. Biophys. Acta 396, 210 219 5 James,S. (1980) Parasitology 80, 313 322 6 Wang, C.C. et at. (1975) J. Protozool. 22, 564 568 7 Ivanetich, K.M, and Santi, D.V. (1990) Exp. Parasitol. 70, 367 371 8 Smith, C.K. and Galloway, R.B. (1983)J. Parasitol. 69, 666 670 9 Augustine, P.C. et al (1987) Poultry Sci. 66, 960 965

I0 Weppelman, R.M. et al. (1977) Poultry Sci. 56, 1550 1559 I I McDougald, L.R. et al (1987) Avian Dis. 3 I, 302-308 12 Chapman, H.D. (1984) • Parasitenkd 70, 437 441 13 Joyner, L.P. and Norton, C.C, (1978) Parosit ology 76, 369 377 14 Long, P.L. et al. (1985) Poultry Sci 64, 2403 2405 15 Chapman, H.D. (1989) Res. Vet, Sci 47,

125 128 16 Jeffers,T.K. and Challey, J.R. (I 973)J. ParasitoL 59, 624 630 17 Shidey, M.W. and Long, P.L. (1990) in Coccidiosis of Man and Domestic Animals

(Long, P.L, ed.), pp 321 341, CRC Press

H. David Chapman is at the Department of Poultry Science, University of Arkansas, Fayettevitle, AR 7270 t 120 I, USA.

Drug Resistance in Schistosomes D. Cioli, L Pica-Mattoccia and S. Archer Drug resistance in schistosomes is con fined essentially to compounds of the hycanthone/oxamniquine family, since no documented case of resistance has so far been reported for the widely used drug praziquantel. The availability of strains of Schistosoma mansoni that are resistant to hycanthone and oxamniquine has permitted a detailed genetic and biochemi cal study of the mechanism of action of these compounds. Drugs must be activated by enzymatic esterification and this ultimately results in the production of an electrophilic moiety capable of alkylating DNA and other parasite macromolecules. As reviewed here by Donato Cioli, Livia Pica-Mattoccia and Sydney Archer, resistance is due to the loss of a drug-activating enzyme that is present in sensitive schisto somes and absent in resistant worms and in the mammalian hosts. Further study of this enzyme may yield valuable clues for drug design and for a basic understand ing of parasite metabolism.

Resistance is usually defined as a genetically transmitted loss of sensitivity in a parasite population that was previously sensitive to a given drug. The lack of sensitivity in a previously unexposed population (natural resistance) is often termed tolerance ~, The boundary between the two situations is rather blurred when viewed in molecular terms, since in either case one has to deal with one or more mutations differentiating a given population from another one. In the case of resistance, the mutation manifests itself in time and may have been selected by the drug; in the case of tolerance, the divergence has occurred previously and usually manifests itself in space under the form of different geographical strains subjected only to natural selection. The earliest reports of genetically transmitted differences in drug sus-

ceptibility came from comparisons between different geographical strains. Also, an extension of the 'strain' concept is implicit in the observation that different species within the genus Schistosoma have different susceptibilities to schistosomicides. Miscellaneous Schistosomicides

The efficacy of antimonial compounds against experimental infections with a Japanese strain of S. japonicum was shown by Hs0 et al. to be lower than that against infections with Chinese, Formosan or Philippine strains2. Direct attempts to demonstrate, in the laboratory, the development of resistance to this class of compounds gave inconclusive results 3. As to niridazole, it was shown that its efficacy in mouse infections was higher against a Puerto Rican strain of S. mansoni than against a Tanzanian strain 4. A Kenyan strain of S. mansoni (KMN-I) showing low susceptibility to niridazole has been reported S, but no account of experimental work with this strain has appeared. Antimonials and niridazole are obsolete drugs, but the existence of strain-related variations in the efficacy of these compounds may be taken as an indication that schistosomes have at least the potential of developing resistance to a wide variety of chemicals. Metrifonate is effective against S. hematobium and virtually ineffective against S. mansoni and S. japonicum 6. Attempts to mutagenize schistosomes by exposing infected snails to ethyl methane sulfonate appeared to yield schistosomula with increased survival, in vitro, in the presence of metrifonate, but these experiments were not carried out beyond a very preliminary stage 1.

Praziquantel has a very good record of uniformly high efficacy against all schistosome species and strains. A recent report of an exceptionally low cure rate in a Senegalese focus is being closely watched (F.F. Stelma et al., abstract*) but no documented case of praziquantel resistance has yet appeared in the literature. Although the mechanism of action of praziquantel is still unclear 8, there must be several possible mutations in the schistosome genome which could render the drug ineffective. Therefore, it is important to be prepared with alternative drugs, and it is important that hypothetical resistant mutants be carefully isolated since they could provide valuable tools in the study of drug mechanisms~. The largest amount of information on drug resistance in schistosomiasis is concerned with drugs of the hycanthone/oxamniquine family. The inefficacy of these compounds against S. japonicum can be mentioned as a preliminary evidence for 'tolerance' in at least one species of schistosomes. Also, a broad trend is generally recognized for a lower oxamniquine susceptibility of African S. mansoni in comparison with South American strains6.

HycanthonelOxamniquine Resistance

In 1955, GOnnert and Vogel clearly showed that mice infected with an Egyptian strain of S. mansoni were less sensitive to lucanthone than were mice infected with a Liberian strain 9. However, it was not until 1971 that the first documented report of true drug resist

'kVI European Multicolloquium of ParasitoLogy (1992) The Hague, The Netherlands © 1993. Flsevier Science Publishers l i d (UK}