Effect of Amprolium on Production, Sporulation, and

Effect of Amprolium on Production, Sporulation, and Infectivity of Eimeria Oocysts Author(s): M. D. Ruff, R. Garcia, M. B. Chute and T. Tamas Source: Avian Diseases, Vol. 37, No. 4 (Oct. - Dec., 1993), pp. 988-992 Published by: American Association of Avian Pathologists Stable URL: http://www.jstor.org/stable/1591904 Accessed: 20-12-2015 00:07 UTC REFERENCES Linked references are available on JSTOR for this article: http://www.jstor.org/stable/1591904?seq=1&cid=pdf-reference#references_tab_contents You may need to log in to JSTOR to access the linked references.

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AVIANDISEASES37:988-992, 1993

Effect of Amprolium on Production, Sporulation, and Infectivity of Eimeria Oocysts M. D. Ruff,A R. Garcia,B M. B. Chute,A and T. TamasB AProtozoanDiseases Laboratory,Livestock& PoultrySciences Institute, ResearchService, United States Departmentof Agriculture-Agricultural Beltsville, AgricultureResearchCenter-East,Beltsville, Maryland20705 BMerck& Co., Inc., P.O. Box 2000, Rahway,New Jersey 07065 Received 1 February1993 SUMMARY.Amproliumreduced the number of oocysts shed by Eimeria acervulina, E. maxima, E. necatrix, and a mixtureof susceptible strainsof E. tenella. Sporulationof oocysts from medicated chickens was reduced compared with that of oocysts from unmedicated chickens. Sporulationwas reduced by levels of 0.0250%amproliumfor E. acervulina and by levels of 0.0060%for E. maxima and the susceptible E. tenella. Not enough oocysts were recovered to measure sporulationof E. necatrix. Sporulationreduction was not affectedby the method of administrationof amprolium (feed or water), except with E. acervulina, for which fewer oocysts sporulatedwhen 0.0120%amproliumwas added in the drinkingwater than when 0.0125%amproliumwas added to the feed. Conversely,amproliummedication E. tenella.Whenfed to unmedicated had no effecton the sporulationof an amprolium-resistant chickens, those oocysts from amprolium-medicatedchickens that did sporulate were as infective as oocysts recovered from unmedicated chickens. RESUMEN.Efecto del amprolio sobre la produccion, esporulacion e infectividadde los oocistos de Eimeria. El amprolio redujo la eliminaci6n de oocistos de Eimeria acervulina, E. maxima, E. necatrix y una mezcla de cepas susceptibles de E. tenella. Laesporulaci6n de los oocistos de los pollos en tratamientose redujo, comparadacon la observadaen los oocistos de los pollos que no recibieron tratamiento.Estareducci6n fue producidapor el amprolioa niveles de 0.025%paraE. acervulina, y niveles de 0.006%paraE. maximay paralas cepas susceptibles de E. tenella. No se aislaronsuficientes oocistos paramedir la esporulaci6n de E. necatrix. Lareducci6n en la esporulaci6n no se afect6 por el metodo de administraci6ndel amprolio (en el aguao el alimento) excepto con E. acervulina, parala que se encontr6menorcantidad de oocistos esporulados cuando se adiciono amprolio a niveles de 0.012%en el agua de bebida que cuando se adicion6 amprolioa niveles de 0.0125%en el alimento.Contrariamente, no hubo efecto de la medicaci6n con amproliosobre la esporulaci6n de las cepas E. tenella resistentes al amprolio. Los oocistos obtenidos de pollos tratadoscon amprolio tuvieron la misma infectividadcuando fueron suministradosa pollos no tratadoscon amprolio,que los oocistos obtenidos de pollos no medicados. There are several reports in the literature that amprolium medication in feed inhibits sporulation of coccidial oocysts of species such as Eimeria acervulina and E. maxima (3,6). Mathis and McDougald (4,5) found that strains of E. acervulina that developed resistance to amprolium did not demonstrate reduced sporulation. There are no reports to show whether amprolium administered in drinking water, rather than feed, would have the same effect. Amprolium had no effect on the sporulation of oocysts of E. brunetti (3) or E. tenella (1). Effects of amprolium on E. necatrix oocysts have

not been studied. Ruff et al. (7) showed that oocysts recovered from arprinocid-medicated chickens were less infective when given to unmedicated chickens than were equivalent numbers of oocysts recovered from birds that did not receive medication. In light of these results, it is reasonable to look at 1) the effects of amprolium on the sporulation and infectivity of several other strains of coccidia, 2) the loss of sporulation inhibition upon the development of drug resistance in species other than E. acervulina, and 3) effects of medication on the subsequent infectivity of oocysts. The present study

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989

Effect of amprolium on Eimeria

all birds were inoculated with coccidia at the optimum dosage determined above for the species being studied, as determined from the titration trial. Feces were collected during days 5-8 PI, except for the group given E. necatrix, from which feces were collected during days 6-9 PI. Total oocyst output for the 4 days was counted by group using the McMaster chamber method. A portion of the fecal material was placed in petri dishes and allowed to sporulate for 72 MATERIALS AND METHODS hours or more at room temperature until sporulation Birds. Whiteleghorncockerelswere obtainedfrom was complete. This petri-dish method allowed for sufa commercialhatchery(MetzHatchery,Belleville, Pa.) ficient diffusion of oxygen into the liquid to provide at 1 day of age. All birds were kept in wire-floored maximum possible sporulation. The sporulation perbrooder units until 3 weeks of age, at which time the centages were determined microscopically on the first experimental treatment began. Birds older than 3 100 oocysts seen. The remaining portion of the fecal weeks were kept in wire-flooredcages. Feed andwater samples was aerated to promote sporulation, and oowere provided ad libitum. cysts were harvested for studies on infectivity of the Feed. Unmedicated broiler starter(24%protein) oocysts. obtainedfroma commercialfeed mill (SouthernStates, Infectivity of sporulated oocysts. When enough Upper Marlboro,Md.) was fed in all of the experi- oocysts were recovered from chickens within a treatments. ment group, the infectivity of the sporulated oocysts Medication. Amprolium (supplied by MSD was tested. Oocysts within a medication level and AGVET,Rahway,NJ., lot number KSC-337)was add- species were pooled for these studies. Sufficient numed to the feed at levels of 0.0060%,0.0125%,and bers of oocysts were recovered for testing infectivity 0.025%.Feed was assayedto ensure thatpropermed- of E. tenella at a dose of 3.5 x 104 sporulated oocysts/ ication levels were present. Liquid amprolium was bird and of E. acervulina at 1 x 106 sporulated ooused in the drinking water at levels of 0.0060%, cysts/bird. Unmedicated, weight-randomized white 0.0120%,and 0.0240%. leghorn cockerels were inoculated with oocysts, four Coccidia. The species of coccidia and strainsused groups of five chicks each for each group of oocysts for each species were: E. acervulina, a mixtureof LS being tested. Oocysts passaged from the unmedicated (laboratorystrain)#2 and LS#84; E. maxima, LS#68; control group were used as control inoculum. There E. necatrix, LS #52; and E. tenella, a mixture of LS were also four uninoculated control groups in the #10, LS#24, and FS (field strain) #80. In addition, an experiment. At 6 days PI, birds were weighed and killed. Weight gain, feed-conversion ratio (E. tenella amprolium-resistant strain of E. tenella (RS-35; LS#18) was obtained from MSDAGVET. only), and lesion scores were measured (0 = no leInoculation dosages. Titrationswere made of oo- sions, 4 = most severe). Additional studies on infeccystproductionat three inoculationdosages and three tivity in unmedicated chicks using oocyst production medication levels to determine the optimum inocu- as the variable were conducted using susceptible E. lation dosage forthe sporulationexperiments.Twelve tenella at inoculation dosages of 1 x 103 sporulated groups of two white leghorn cockerels each were oocysts/bird and E. acervulina and E. maxima at 2 placed on each of the feed medication levels of am- x 103 sporulated oocysts/bird. Not enough E. necaprolium listed above. Two days later, three groups trix oocysts were recovered from birds at any drug fromeach medication level were inoculatedwith one level for any type of infectivity study. Results were of the fourspecies of coccidia.Eachof the threegroups analyzed using analysis of variance, and differences received a differentlevel of oocysts: 1 x 105, 1 x 106, in means were tested with Duncan's multiple range or 2 x 106 of E. acervulina; 2 x 104, 2 x 105, or 4 x test. 105 of E. maxima or E. necatrix; and 1 x 104, 1 x

examined the sporulation of four species of coccidia following exposure to three different levels of amprolium in the feed or water. When sufficient sporulated oocysts were recovered, they were given to unmedicated chicks to determine relative infectivity.

105, or 2 x 105of E. tenella. Feces were recovered from day 6 through day 9 PI. Total oocyst output for the 4 days was counted for each group. Sporulation of oocysts with amprolium medication. Five experimentswere run, one for each of the four species of coccidia plus the amprolium-resistant E. tenella. In each experiment, 140 white leghorn cockerels were allotted into 28 groups of five birds each at 19 days of age. Fourgroupswere placed on each of the six amprolium levels (three in feed, three in water) indicated above. The remainingfour groups were unmedicated controls.At 21 daysof age,

RESULTS The dosages selected from the titration studies as ones that seemed to give sufficient oocysts for the sporulation studies were 2 x 106 sporulated oocysts/bird for E. acervulina, 2 x 105 for E. maxima, and 1 x 105 for E. tenella. Too few oocysts to count were obtained with E. necatrix and amprolium so the medication, highest dose of 2 x 105 sporulated oocysts/bird was selected for that species.


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M. D. Ruff et al.

Table 1. Number of oocysts recovered from the feces of chickens given various levels of amprolium. Oocysts/bird (x 106) % Amprolium in water

% Amprolium in feed 0

Species E. E. E. E.

acervulina maxima necatrix tenella (susceptible)

0.0060

89 5.0 10.2 77

37 1.3 TFTC 0.4

0.0125 49 TFTCA TFTC 0.2

0.0250

0.0060

0.0120

0.0240

25 TFTC TFTC TFTC

46 2.4 TFTC 0.5

48 TFTC TFTC 0.04

13.6 TFTC TFTC TFTC

ATFTC= too few to count.

Table 2. Sporulation of oocysts recovered from chickens given various levels of amprolium.

Sporulation(%)A % Amprolium in feed 0

Species E. acervulina

% Amprolium in water

0.0060

0.0125

0.0250

0.0060

0.0120

0.0240

57c TFTC TFTC TFTC 90a

88a

66c

37b

51b

TFTC

TFTC

77b TFTC TFTC TFTC 89a

85th 83:'

84ab

E. maxima

54h

82ab 45b

E. necatrix E. tenella (susceptible) E. tenella (resistant)

72 77-' 91;'

TFTC 21c 91'

TFTC 32b 90a

34b

39b

87a

84a

ASporulation based on 100 oocysts. TFTC = Too few to count for sporulation. Within a species, values followed by a common lower-case superscript are not significantly different (P > 0.05).

Table 3. Infectivity of E. tenella oocysts in unmedicated chickens. Oocysts recovered from chickens given various levels of amprolium.A % Amprolium for initial passage 0 (uninoculated) 0 (inoculated) 0.0060 0.0125 0 (inoculated) 0.0060 0.0125 0.0250

StrainB

Weight gain (g)

S S S R R R R

934 ? 29ab 987 ? 44a 832 ? 52bc 910 ? 52ab 663 ? 33d 704 ? 25cd 736 ? 57cd 782 ? 47cd

Feed/Gain 1.92b 1.98b

2.18ab 1.98b 2.47a 2.41a 2.49a 2.15ab

Lesions scoresc Ob

1.92 1.83 1.83 1.92 2.00 1.92 1.83

? 0.08a ? 0.10a ? 0.10a

? ? ? ?

0.08a 0.00a 0.08a 0.20a

AWithin a column, values followed by a common lower-case superscript are not significantly different (P > 0.05). Each value is the mean ? SEM for 20 chicks. BS= Mixture of susceptible strains; R = Resistant strain. cSee Materials and Methods for scoring method.

Medication with amprolium reduced the number of oocysts recovered (Table 1). Medication did not reduce the number of oocysts recovered with the resistant E. tenella (results not shown). Too few oocysts to count were recovered from chickens medicated with 0.0250%

amprolium when given the susceptible E. tenella; 0.0125% when given E. maxima; and all levels when given E. necatrix. There were essentially no differences in oocyst recovery between amprolium in feed or drinking water. Sporulation of E. maxima and the susceptible


991

Effectof amproliumon Eimeria Table4. Infectivityof E. acervulina oocysts in unmedicated chickens. Oocysts recovered from chickens given various levels of amprolium.A %Amproliumfor initial passage

Weight gain (g)

0 (uninoculated)

922 817 840 848 853

0 (inoculated) 0.0060 0.0125 0.0250

? 9:1 ? 1h +? 8b ? 122b ? 10b

passed in unmedicated birds (Table 5). Infectivity was similar, irrespective of the method of amprolium medication.

Lesion scoresB

DISCUSSION

Oh 2.00 ? 0.0a 2.00 ? 0.0a 2.00 ? 0.0a 2.00 ? 0.01

AWithina column, values followed by a common lower-case superscriptare not significantlydifferent (P> 0.05). Each value is the mean ? SEM for 20 chicks. BSeeMaterialsand Methods for scoring method. E. tenella was reduced in feces from chickens given amprolium, even at a level of 0.0060% (Table 2). Sporulation of E. acervulina was reduced at amprolium levels of 0.0250% in the feed or 0.0120% in water. Sporulation of the resistant E. tenella was not affected, even by levels of amprolium as high as 0.0250%. When the sporulated oocysts were inoculated into unmedicated chickens to test infectivity, the oocysts of the resistant E. tenella and E. acervulina from amprolium-medicated chickens were as infective as those passed in unmedicated chickens (Tables 3 and 4) based on depressed weight gain, feed gain (E. tenella only), and lesion scores. The same inoculation dose used with the resistant E. tenella failed to produce any weight depression with the susceptible E. tenella. Based on oocyst production, the infectivity of E. maxima, E. acervulina, and the susceptible E. tenella from birds medicated with amprolium was as great as the infectivity of the parent strains

Based on the effects on the number of oocysts produced by the strains of coccidia used in the present study, amprolium was highly efficacious against E. maxima, E. necatrix, and susceptible E. tenella, moderately effective against E. acervulina, and ineffective against a resistant E. tenella. Amprolium reduced sporulation of all species except for the resistant E. tenella strain. Amprolium reduced sporulation of E. acervulina, E. brunetti, and E. maxima from Weybridge, England (3). Conversely, amprolium had no effect on the sporulation of E. tenella from Japan (1). Ball et al. (2) showed that the reduction in sporulation of E. acervulina was accompanied by large numbers of small wallforming bodies in the macrogametes, leading to abnormal oocyst wall formation. Some other anticoccidials, such as arprinocid (7,8) and dinitolmide (3,4,6), markedly reduce sporulation of recovered oocysts. Other anticoccidials, such as monesin, salinomycin, and lasalocid, do not reduce sporulation (1). Mathis and McDougald (4) first reported that resistance of E. acervulina to amprolium and dinitolmide was accompanied by loss of the drug's effect on sporulation. They later showed (5) a similar loss of the inhibition of sporulation when resistance to the same two compounds was produced in the laboratory. The present study shows that a loss of inhibition also occurs with amprolium resistance in E. tenella. Ruff et al. (7) reported that oocysts harvested

Table 5. Numberof oocysts recovered from the feces of unmedicated chickens. The source of the oocysts for the inoculation was chickens given various levels of amproliumin the feed or water as indicated. Oocysts/bird (x 106)A

%Amproliumin feed of source birds Species E. tenella E. acervulina E. maxima

%Amproliumin water of source birds

0

0.0060

0.0125

0.0250

0.0060

0.0120

0.0240

1.8 57.2 3.2

2.2 68.0 0.9

0.7 61.0 1.8

ND 55.3 2.4

1.2 47.1 2.4

ND 57.2 1.7

ND 82.0 3.0

AND = not done.


M. D. Ruff et al.

992

from chickens given arprinocid were less infective than those from unmedicated chickens. No such effect on subsequent infectivity was found in the present study. Similarly, Arakawa et al. (1) noted no difference in infectivity between oocysts recovered from unmedicated chickens and those recovered from clopidolmedicated chickens. REFERENCES 1. Arakawa,A., Y. Tanaka,E. Baba,and T. Fukata. Effects of clopidol on sporulation and infectivity of Eimeriatenella oocysts.Vet. Parasitol.38:55-60. 1991. 2. Ball, S. J., R. M. Pittilo, C. C. Norton, and L. P. studies of the effect of amproJoyner. Ultrastructural lium and dinitolmide on Eimeriaacervulinamacrogametes. Parasitol.Res. 73:293-297. 1987. 3. Joyner,L.P.,and C. C. Norton. The anticoccidial effectof amprolium,dinitolmideandmonensinagainst Eimeriamaxima, E. brunetti, and E. acervulinawith

particular reference to oocyst sporulation. Parasitology 75:155-164. 1977. 4. Mathis, G. F., and L. R. McDougald. Effect of amprolium and dinitolmide on sporulation of oocysts of field isolates of Eimeria acervulina. Parasitology 83: 281-284. 1985. 5. Mathis, G. F., and L. R. McDougald. Experimental development of resistance of amprolium or dinitolmide in Eimeria acervulina and its effect on inhibition of sporulation of oocysts. J. Parasitol. 67: 956-957. 1981. 6. Norton, C. C., and L. P. Joyner. Effects of some anticoccidial drugs on sporogony of Eimeria species. In: Avian coccidiosis. Br. Poult. Sci., Edinburgh, Scotland. pp. 435-444. 1978. 7. Ruff, M. D., W. I. Anderson, and W. M. Reid. Effect of the anticoccidial arprinocid on production, sporulation, and infectivity of Eimeria oocysts. J. Parasitol. 64:306-311. 1978. 8. Tamas, T., G. Olson, D. A. Smith, and B. M. Miller. Effect of 6-amino- (substitute benzyl) purines on oocyst sporulation. Poult. Sci. 57:381-385. 1978.
