EXPERIMENTAL

Eimeria

PARASITOLOGY

tenella:

The

42, 56-66

Selection

R. M. WEPPELMAN, Merck

Sharp

G Dohme

(1977)

and

Frequency

J. A. BATTAGLIA,

Research Laboratories,

(Accepted

for publication

Rahway,

of Drug-Resistant

Mutants

AND C. C. WANG New

Jersey

07065,

U.S.A.

13 October 1976)

WEPPELMAN, R. M., BATTAGLU, J. A., AND WANG, C. C. 1977. Eimeria tenella: The selection and frequency of drug-resistant mutants. Experimental Parasitology 42, 56-66. Mutants of Eimeria tenella resistant to amquinate or to glycarbylamide were isolated by serially passing sporulated oocysts three times through chickens medicated with the commercially recommended level of these anticoccidial compounds. The frequency of mutants resistant to glycarbylamide was found to be 2.4 X 10” per wild-type oocyst, while the frequency of mutants resistant to amquinate was found to be 5.8 X lo-’ per wild-type oocyst. The same procedure failed to yield mutants resistant to amprolium, nicarbazin, robenidine or monensin, suggesting that mutants resistant to these are all less frequent than 7.5 X 10-O per wild-type oocyst. INDEX DESCRIPTORS: Eimeria tenella; Parasitic protozoa; Coccidia; Coccidiosis; Chickens; Drug-resistant mutants; Amprolium; Amquinate; Glycarbylamide; Monensin; Nicarbazin; Robenidine.

The use of suboptimal drug levels was implicitly based on the hypothesis that Traditionally drug-resistant coccidia organisms could be adapted to tolerate have been isolated by the procedure first normally toxic concentrations of these used by Cuckler and M’alanga (1955) to chemicals and the adaptation was thought isolate strains of Eimeria tenella and to be genetic, enzymic, or physicochemical Eimeria acervulina resistant to sulfaquinoxaline. These authors serially passed 8coccidi.a (Cuckler and Malanga 1955). Subsequent through chickens medicated with sub- authors have noted that resistance persists optimal drug levels, which permitted sub- even after the particuIar medication has stantial leakage of oocysts, and after five been withdrawn (Cuckler, McManus, and 1969; Ryley and Belts 1973; or ten such passages, the strains showed Campbell Jeffers 197413) which indicates that resista diminished response to sulfaquinoxaline. ance is heritable and of a genetic nature. Using similar procedures, all of which The number of repetitive exposures to employed suboptimal medication, various suboptimal medication required for the laboratories have isolated coccidia resistant emergence of resistance has traditionally to the quinoline anticoccidials, amquibeen used as an indication of the ease nate (McManus, Campbell, and Cuckler with which resistance to a particular drug 1968) and buquinolate ( McLaughlin 1970)) is acquired. Since different laboratories to glycarbylamide (Bjall 1966; Cuckler, often used different levels of suboptimal McManus, and Campbell 1969), to nicarmedication and different numbers of organbazin ( McLaughlin and Gardiner 1967), was inand to amprolium (McLaughlin and isms for each passage, variability Gardiner 1968 ) . troduced, and the results from these lab56 CopyrIght AU rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0014-4894

Eimeria

tenella:

oratories often did not correlate with each other or with the rat,e at which resistance to a particular drug developed in the field (Ryley and Betts 1973). As an alternative to the hypothesis that resistance results from adaptation, the present communication proposes that popuIations of coccidia became resistant to anticoccidial compounds through the selection of spontaneous resistmant mutants which are present, albeit in very small numbers, even in populations which have never been exposed to the drug. If resistance results from selection rather than adaptation, then the drug serves only to select pre-existing mutants and it should prove possible to isolate these mutants by selection with the recommended level of the drug rather than with a suboptimal level. The only conceivable case in which a drug might induce resistance as well as select for resistance would be if the drug possessedboth mutagenic and anticoccidial activities. Since mutagenicity has not been attributed to any marketed anticoccidial compound, this would seem unlikely. Thus most drugresistant coccidia apparently result from spontaneous mutations whose occurrence is independent of the drug. The present communication describes the isolation of E. tenelZa mutants resistant of glycarbylamide or to amquinate after selection with the optimal levels of these drugs. The isolation procedure, which was based on the fluctuation test of Luria and Delbriick ( 1943), p ermitted an evaluation of the frequency of resistant mutants in the coccidial population. This was accomplished by dividing the wild type population from which mutants were to be selected into numerous groups containing equal numbers of sporulated oocysts. Each group was then passed through five chickens receiving optimal medication. The progeny from groups which reproduced were passed a second time through optimally medicated ‘chickens, and the descendants of groups reproducing during the second

57

DRUG RESISTANCE

passage were passed #a third time, again through chickens receiving optimal medication, to verify that they were drug resistant. The number of resistant mutants per group was then calculated with the Poisson distribution from the fraction of the groups which failed to produce resistant descendants and which by inference had not contained resistant mutants. MATERIALS

AND METHODS

Coccidial Strain Eimeria tenella, Merck strain 18, was used for all experiments. This strain, descended from a single sporulated oocyst from a field isolate, is not known to be resistant to any commercially available anticoccidial drug. The routine passage and purification of oocysts were performed by the procedures of Wagenback, Challey, and Burns (1966). Chickens Hubbard x Hubbard pullets, 3-4 weeks of age and fed lad libitum on standard broiler ration, were used for all drugresistance experiments. Infection was accomplished by inoculation per OS. When resistant mutants were isolated, chickens infected with ‘different coccidial isolates were caged separately to avoid cross-contamination of the isolates. Partial Purification of Oocysts For the isolation of resistant mutants, five pairs of infected ceca were harvested and ‘added to 100 ml of 0.1 M NaOH. The mixture was dispersed with a Waring Blendor and was then incubated at 29 C with vigorous shaking for 2 days to permit sporulation. The mixture was then ‘centrifuged at 104g for 10 min and the supernatant was decanted. The pellet containing oocysts was resuspended in 20 ml of 5% sodium hypochlorite, and was incubated at 0 C for 10 min to permit the digestion of contaminatiing host tissue. Eightly milliliters of water was then added and the

58

WEPPELMAN,

BATTAGLIA

Selection Passage Five medicated chickens were used to pass each group of 5 X 10’ sporulated wild-type oocysts. Groups producing fewer than 8.5 X lo3 sporulated oocysts were discarded. .I, Enrichment Passage Progeny from the groups remaining were passed ,&rough five medicated chickens. Groups producing fewer than 6 X lo4 sporulated ‘oocysts during this passage were discarded. \1 Test Passage Four medicated chickens were each infected with 104, 5 X 104, or 2.5 X lo6 sporulated oocysts descended from a group during the preceding passage. Weight gains, pathology, and oocyst production were determined. FIG. 1. Procedure for isolation tants of Eimeria tenelk

of resistant mu-

mixture was centrifuged as described above. The pellet was resuspended in 100 ml of water and the centrifugation was repeated. This final pellet was resuspended in 5.0 ml of water and the sporulated oocysts were counted with a hemocytometer. The Isolation Procedure The isolation of resistant mutants was accomplished in three sequential passages as shown in Fig. 1. Chickens were medicated with the commercially recommended level of the anticoccidial compound in the diet for all three passages. The first passage, the selection passage, was performed to select from a large number of drug-sensitive organisms any drugresistant mutants which might be present. In preparation for this passage, a population of wild-type E. tenella which had never been exposed to anticoccidials was divided into groups of 5 x lo6 sporulated oocysts each. This rellatively large number of sporulated oocysts was used to increase the probability that each group would contain a resistant mutant. Each group was passed by inocullating five chickens with 1 x lo6 sporulated oocysts each. After 8 days, the five pair of ceca, containing the

AND

WANG

combined progeny of the group, were harvested as described above and their combined oocyst content was determined. The use of 5 ~10~ sporulated oocysts and five chickens to pass each group was an arbitrary compromise between the desirability of exposing large numbers of parasites to selection and the availability of animal facilities. It would probably be desinable both statistically and biologically to use more groups, each containing fewer oocysts than were used here. The second passage,the enrichment passage, was performed to increase the number of resistant mutants among the progeny produced by the various groups during the selection passage. For this passage, the progeny from those groups which had produced detectable progeny during the selection passage were used to inoculate five chickens receiving the appropriate medication. The inoculum per chicken was 5 x lo4 or less, depending on the number of sporulated oocysts produced during the selection passage. The inocula were restricted to a maximum of 5 x lo4 in order to minimize the possibility of isolating a resistant mutant which, through random mutation, had been generated during the selection passage. The purpose of this isolation procedure was to determine the frequency of resistant mutants in the inocula used for the selection passage, and mutants generated during the selection passage would artificially inflate the result. Restricting the inocula for the enrichment passage to one twentieth of that used for the selection passage ensured that the probability of isolating a mutant generated during the selection passagewas only onetwentieth that of isolating a mutant present at the start of the selection passage. Eight days postinfection, the five chickens were sacrificed, their ceca were harvested as described above, and the combined sporulated oocyst content was determined. The third sequential passage. the test passage, was performed to test the hy-

Eimeriu

tenella:

DRUG

TABLE

59

RESISTANCE

I

Results of the Eirneriu tenetlu Selection Passage Medication

Percentage of fatalities

0.006% Glycarbylamide

Eimeria tenella: the selection and frequency of drug-resistant mutants.

EXPERIMENTAL Eimeria PARASITOLOGY tenella: The 42, 56-66 Selection R. M. WEPPELMAN, Merck Sharp G Dohme (1977) and Frequency J. A. BATTAG...
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