Effect of Feed Additive Antibiotics on Chickens Infected with Eimeria tenella O. O H E AND A. ARAKAWA
Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 2-1-6, Kashima-cho, Osaka 532, Japan
Yodagawa-ku,
(Received for publication September 17, 1974)
POULTRY SCIENCE 54: 1008-1018, 1975
INTRODUCTION
S
EVERAL workers reported changes in the bacterial flora of intestinal tract of chickens as a result of coccidia infection. Johansson and Sarles (1948) reported increase of Clostridium perfringens and decrease of lactobacilli and enterococci in the ceca of chickens infected with Eimeria tenella. Similar findings were reported by Lafont (1966) and Bradley and Radhakrishnan (1973). Hein and Timms (1972) found significant increase of C. perfringens and Escherichia coli in small intestine and ceca of chickens infected with E. brunetti. Recent studies confirmed the increase of C. perfringens in the ceca of chickens infected with E. tenella and of coccidiostat-treated chickens infected with coccidiostat-resistant E. tenella, and showed that the increase was suppressed by supplementing antibiotics in the feed (Arakawa and Ohe, 1975). Antibiotics found to be effective
were thiopeptin at 2 mg./kg. of feed, bacitracin at 20 mg./kg., penicillin at 12 mg./kg., and chlortetracycline at 22 mg./kg. The purpose of the present experiments was to study what effect these antibiotics given via the feed would have on body weight, mortality and oocyst production in chickens infected with E. tenella and in coccidiostattreated chickens infected with coccidiostatresistant strains of E. tenella.
MATERIALS AND METHODS Birds. Day-old broiler chicks, Arbor AcreVantress crossbreed, were obtained from a commercial hatchery and were reared in conventional electrically heated battery brooders until use. They were caged in batteries with raised screen bottoms in a room of a heated building, ventilated and constructed to prevent excessive temperature changes.
1008
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ABSTRACT Six experiments were carried out to study the effect of feed additive antibiotics on body weight, mortality and oocyst production in chickens infected with coccidiostat-susceptible or -resistant strain of E. tenella. Levels of antibiotic and coccidiostat in fed (mg./kg.) were: thiopeptin, 2; zinc bacitracin, 20; penicillin, 12; chlortetracycline, 22; amprolium plus ethopabate, 125 plus 8; clopidol, 125. All experiments included 7 groups; 2 of these groups were uninfected and infected controls, and the 5 remaining groups were all infected and given diet containing antibiotic, amprolium plus ethopabate, antibiotic and amprolium plus ethopabate, clopidol, or antibiotic and clopidol. Chickens in each group were fed respective diet beginning one day prior to coccidia exposure. In two experiments, infection with a coccidiostat-susceptible strain resulted in severe clinical coccidiosis in chickens on the basal ration and on thiopeptin-diet, but dietary thiopeptin prompted recovery of body weight. In one experiment where chickens were infected with a strain resistant to amprolium plus ethopabate and clopidol, birds on dietary thiopeptin attained higher body weight than birds on the basal ration. In three experiments when a strain resistant to amprolium plus ethopabate was inoculated, birds given the basal ration, bacitracin, penicillin, chlortetracycline, or amprolium plus ethopabate diet developed cecal coccidiosis. Chickens on ration containing antibiotic alone attained higher body weight than chickens on the basal ration. Combination of antibiotic and amprolium plus ethopabate resulted in higher weight attained than amprolium plus ethopabate alone. Clopidol suppressed development of coccidiosis, and the combination of antibiotic and clopidol resulted in higher gains than in clopidol alone.
ANTIBIOTIC FEEDING IN COCCIDIOSIS
Diet. The basal ration used was reported in the previous study (Arakawa and Ohe, 1975). Medicated feed was prepared by mixing the basal ration with a measured amount of commercial premix. Levels of antibiotic and coccidiostat in feed (mg./kg.) were: thiopeptin, 1 2; zinc bacitracin, 2 20; procaine penicillin G, 3 12; chlortetracycline hydrochloride, 4 22; amprolium plus ethopabate, 5 125 plus 8; and clopidol, 6 125.
Coccidial Challenge. Coccidiostat-susceptible strain of E. tenella was maintained by Dr. K. Tsunoda of the National Institute of Animal Health in Tokyo and supplied to this laboratory. This strain was used in Experiments 1 and 2. Two coccidiostat-resistant strains of E. tenella were obtained from litter
1. 2. 3. 4. 5. 6.
Fujisawa Pharmaceutical Co., Ltd., Osaka. Nippon Kayaku Co., Ltd., Osaka. Taito Pfizer Co., Ltd., Tokyo. Takeda Chemical Industries, Osaka. Marupi-Merck Sharp and Dohme, Osaka. Tanabe Co., Ltd., Osaka.
samples from local broiler houses, separated by a single oocyst method, and propagated through chickens under strictly isolated conditions. These strains were tested previously by the methods described by McManus et al. (1968). One strain used in Experiment 3 was resistant to amprolium plus ethopabate (the anticoccidial index = 130 at 125 plus 8 mg./kg. in the feed) and clopidol (the index = 134 at 125 mg./kg.), and another strain used in Experiments 4, 5, and 6 was resistant to amprolium plus ethopabate (the index = 121 at 125 plus 8 mg./kg.). Fresh oocyst cultures were prepared routinely from donor chickens 7 to 8 days after infection. Chickens in infected groups were each given an oral inoculation of 30,000 sporulated oocysts. Daily oocyst counts were made starting 7 (Experiments 3,4,5, and6)or 8 (Experiment 1) days after infection. Fecal material was collected from each cage and was well mixed to obtain estimates of oocyst production. Appropriate aliquots were taken, diluted, and placed on a plankton counting plate (Tsunoda and Ishii, 1971). Birds that were withdrawn during the Experiment 1 and those that died were necropsied for gross pathological observations. Cecal lesions were scored using a 0 to 4 system as described by Johnson and Reid (1970). Statistical Methods. Analysis of variance and Duncan's new multiple range test (P = 0.05) were performed for statistical evaluation of differences in body weight among 7 groups (Steel and Torrie, 1960). Experiment 1. Seventeen-day-old chickens, males weighing 280 to 285 g. and females, 270 to 275 g., were used. Rations containing thiopeptin and coccidiostats were fed consecutively for 14 days. Infected groups were exposed to coccidiostat-susceptible strain of E. tenella. Seven days after infection 10 birds, 5 males and 5 females, were randomly selected from each group and necropsied for macro-
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Effect of Antibiotics on Chickens. Six experiments were conducted. In each experiment, a total of 140 chickens were divided into uniform groups of 7 consisting of 10 males and 10 females each on the basis of body weight. The groups consisted of those infected fed a ration containing either antibiotic, amprolium plus ethopabate, antibiotic and amprolium plus ethopabate, clopidol, or antibiotic and clopidol. The two controls were an infected, unmedicated group and an uninfected, unmedieated group. The birds in each group received respective ration beginning one day before coccidia exposure and were fed continuously until the termination of experiments. Water was available ad libitum. Birds were individually weighed daily during the first 6 or 7 days and weekly thereafter, excepting Experiment 6 that terminated 18 days after infection.
1009
416
485 ab 515 a
479 abc
469 ab 467 ab
513 a
None
455 a b c
494 ab
421 b c
421 b c 475 a
450 "°
2 125 8 2 125 8 125 2 125
Female
None
thiopeptin amprolium ethopabate thiopeptin amprolium ethopabate clopidol thiopeptin clopidol
Male 432=
6 days (10 birds) abc
7)8
abc
705 bc
675 c 808 a
778 a
661 c
722
Male 675 ab 665 ab
97.3 100
596 b
106.5 109.7
658 ab
671 a b 693 a
651 a b 108.1
95.3 108.2
Female
Yo
relativ weight 6 days
13 days (5 birds)
Average live weight (g.)'
'Within each column, any averages having the same superscript are not significantly different at 0.05 2 Days after coccidia infection. 'Average of 8 birds (5 males and 3 females).
Inf. unmed. Uninf. unmed.
Inf. Inf.
Inf.
Inf. Inf.
Group
2
of thiopeptin and coccidiostats on chickens infected with a coccidiostat-suscep
Supplement in feed (mg./kg.)
TABLE 1.—Effect
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1011
ANTIBIOTIC FEEDING IN COCCIDIOSIS
scopical observations with the exception of infected control where only 8 birds (5 males and 3 females) were necropsied. Daily oocyst counts were made in the 5 remaining birds.
Experiment 3. Birds, 14 days old, were used in 21-day experiment. Their weight ranged from 200 to 220 g. for males and 190 to 210 g. for females. Feed supplemented with thiopeptin and coccidiostats was given. Chickens in infected groups were each given an oral inoculation of E. tenella oocysts that are resistant to amprolium plus ethopabate and clopidol.
Experiment 5. The average body weight of the birds on the 21-day feeding trial were males 245 g. and females 235 g. The same strain of E. tenella was used to infect the infected group as in Experiment 4. Experiment 6. Eighteen-day-old chickens, weighing an average of 330 g. for males and 325 g. for females, were used for 19-day study. Feed was medicated with chlortetracycline and coccidiostats. Coccidia inoculated were the same strain used in Experiment 4. RESULTS Experiment 1. Table 1 summarizes the results. Females in uninfected control group were attracted to the feed much slower than
TABLE 2.- -Effect of thiopeptin and coccidiostats on chickens infected with a coccidiostat-susceptible
strain ofE. tenella (Experiment 2)
% re lative w eight
Average live weight (g.)' 6 days 2 Group Inf. Inf. Inf.
Inf. Inf. Inf. unmed. Uninf. unmed.
Feed (mg./kg.) thiopeptin amprolium ethopabate thiopeptin amprolium ethopabate clopidol thiopeptin clopidol
2 125 8 2 125 8 125 2 125
Male 150 194a
Female 132 175b
20 days Male
6 days
20 days
545 606 ab
560a 567"
76.6 100.2
98.1 104.4
Female
189ab
195
575 c
572 a
104.3
101.8
194a 195a
183 ab 175b
628 a 589 >*
548 ab 534"
102.4 100.5
104.4 99.7
None
171
141
593 ^
486 c
84.7
95.8
None
184"
184a
623 a
503 c
100
100
'Within each column, any averages having the same superscript are not significantly different at 0.05 level of probability. 2 Days after coccidia infection.
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Experiment 2. Chickens, 7 days old, ranging from 70 to 95 g. of body weight in both sexes were used. This experiment was designed to observe change in body weight for longer period than in Experiment 1; no chickens were withdrawn except birds that died. Feed containing thiopeptin and coccidiostats was given continuously for 21 days. Coccidia inoculated were the same strain used in Experiment 1.
Experiment 4. Chickens, 14 days old, were used. Feed containing bacitracin and coccidiostats was given for 21 days. Birds in the infected groups were each exposed to E. tenella that are resistant to amprolium plus ethopabate.
thiopeptin amprolium ethopabate thiopeptin amprolium ethopabate clopidol thiopeptin clopidol
2 125 8 2 125 8 125 2 125 791 a
801 a 765 a
919 a
869 a 869 a
395 a 366 bc
4 0 7 ab 403 ab
796 a 790 a
353 cd
388"
348 c
387 b
20 days Female
Male 882 a 890 a
Female 326= 3 5 5 cd
6 days
Male
%
98.1 94.2
90.5
relative weight 6 days 20 82.5 90.9
Inf. 764 a None 345 c 876 a 328 d unmed. 82.3 Uninf. 429 a 924 a 841 a None unmed. 100 1 388 a 'Within each column, any averages having the same superscript are not significantly different at 0.05 2 Days after coccidia infection.
Inf. Inf.
Inf.
Inf. Inf.
Group
2
Average live weight (g.)'
of thiopeptin and coccidiostats on chickens infected with a strain o / E . tenella resistan (Experiment 3)
Supplement in feed (mg./kg.)
TABLE 3.—Effect
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ANTIBIOTIC FEEDING IN COCCIDIOSIS
the birds in other groups. This was reflected in their lower weight on 6 and 13 days after infection. Average weights on 6 days after infection of birds given the basal ration and dietary thiopeptin were significantly (P < 0.05) lower than those receiving coccidiostats. Oocyst discharge in feces was not affected by thiopeptin medication.
Experiment 3. Infection with coccidiostatresistant E. tenella resulted in weight depression in all birds on 6 days after infection (Table 3). Infected chickens receiving the basal feed and dietary thiopeptin were significantly (P < 0.05) lower than the rest of groups. Average weight of chickens given amprolium plus ethopabate was significantly lower (P < 0.05) than those in uninfected control. Birds on dietary clopidol were behind in their weight as compared with uninfected control, but the difference was not statistically significant (P > 0.05). Weights of all infected groups taken on 20 days after infection were lower than those of uninfected control, but there were no statistical differences (P > 0.05) among 7 groups. Infected chickens on medicated diets discharged oocysts comparable to those on the basal ration, but the birds given clopidol diet showed delayed and prolonged discharge of oocysts. Experiment 4. Table 4 contains the results. There were no statistical differences (P > 0.05) in weight on 5 and 20 days after infection
between uninfected group and bacitracintreated infected group. Clopidol prevented an outbreak of coccidiosis, and a combination with bacitracin resulted in better performance than in clopidol alone. Percentages of relative weight of birds on dietary bacitracin were greater than those on the diet without bacitracin. Oocyst counts in infected birds given amprolium plus ethopabate diet were fewer than those in infected control. Experiment 5. Results are presented in Table 5. Body weight of birds in infected control group was the lowest (P < 0.05) among 7 groups on 5 and 20 days after infection. Medication of amprolium plus ethopabate alone did not fully protect from coccidia infection as evaluated by body weight on 5 days after infection and by oocyst production. Feeding the ration containing penicillin and amprolium plus ethopabate resulted in better performance than amprolium plus ethopabate alone. Oocyst discharge was not influenced by penicillin medication. Experiment 6. Results are shown in Table 6. Weights on 5 days after infection of infected birds on the basal ration, on chlortetracycline diet, and amprolium plus ethopabate diet were significantly (P < 0.05) lower than those in uninfected control group. By 18 days after infection, the birds on chlortetracycline diet recovered their weight equal to uninfected control (P > 0.05), whereas infected birds on the basal feed and those on amprolium plus ethopabate diet did not recover. Chickens on feed supplemented with either chlortetracycline alone or combined with the coccidiostats attained greater body weight on 18 days after infection than those given without antibiotic. Clopidol suppressed coccidiosis. Oocyst discharge was not affected by chlortetracycline medication. Gross Pathology. In birds that were withdrawn 7 days after infection in Experiment
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Experiment 2. Body weight on 6 days after infection of the surviving birds given the basal feed and dietary thiopeptin was significantly (P < 0.05) lower than those of the rest of groups (Table 2). Percentage of relative weight indicated that birds fed dietary thiopeptin lost more weight by 6 days after infection than those in infected control, but by 20 days after infection the birds on thiopeptin-diet attained greater body weight than the infected control.
1013
851 a 841 a b
358 ^
None
876 d 939 a
325 b 345 a
347" 344a
892 cd 1,010
342 375 a
835 a b
911bc
332 b
356 ^
845 a
821 b c
845 a 802 c
Female
920 ab 930 a
352" 331 b
20 days
366 a b 365 a b
353 c
20 125 8 20 125 8 125 20 125
None
bacitracin amprolium ethopabate bacitracin amprolium ethopabate clopidol bacitracin clopidol
Male
Female
5 days
Male
100
96.4
98.0 102.2
97.8
102.1 99.0
% relative weight 20 5 days
'Within each column, any averages having the same superscript are not significantly different at 0.05 2 Days after coccidia infection.
Inf. unmed. Uninf. unmed.
Inf. Inf.
Inf.
Inf. Inf.
Group
2
Average live weight (g.)'
of bacitracin and coccidiostats on chickens infected with a strain of E. tenella r (Experiment 4)
Supplement in feed (mg./kg.)
TABLE 4.—Effect
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878 c
394 a
41 5 bc
862 c 960 a
None
387 a 394"
413 bc 431 a
927 b
812
385 a
423 ab
925" 953"
358
383 a 373
412 c 408 c
386
12 125 8 12 125 8 125 12 125
Male
None
penicillin amprolium ethopabate penicillin amprolium ethopabate clopidol penicillin clopidol
Female
5 days
Male
91.9 100
820 bc
98.8 102.0
100
98.2 96.5
5 days
758
833 ab 831 a b
850"
800 c
817bc
Female
20 days
%
relati weig
Within each column, any averages having the same superscript are not significantly different at 0.0 2 Days after coccidia infection.
1
Inf. unmed. Uninf. unmed.
Inf. Inf.
Inf.
Group Inf. Inf.
2
Average live weight (g.)'
of penicillin and coccidiostats on chickens infected with a strain ofE. tenella resista
Supplement in feed (mg./kg.)
TABLE 5.—Effect
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499"
492 be 516 a
534 b
549 a 560 a c
953 c 982 b
l,040 bc l,060 ab
522 a
535"
None
l,010 a
939
992 ab
Female 99 0 ab 922
475 d
1,096 1,121
l,069 a
l,038 b c 1,023c
487 c 475 d
513 c 512°
509 c
22 125 8 22 125 8 125 22 125
18 days
None
chlortetracycline amprolium ethopabate chlortetracycline amprolium ethopabate clopidol chlortetracycline clopidol
Male
Female
5 days
Male
100
93.1
98.4 101.8
97.7
94.6 93.3
5 days
% relativ weigh
'Within each column, any averages having the same superscript are not significantly different at 0.0 2 Days after coccidia infection.
Inf. unmed. Uninf. unmed.
Inf. Inf.
Inf.
Inf. Inf.
Group
2
Average live weight (g.)'
of chlortetracycline and coccidiostats on chickens infected with a strain of E. ten (Experiment 6)
Supplement in feed (mg./kg.)
TABLE 6.—Effect
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1017
ANTIBIOTIC FEEDING IN COCCIDIOSIS
TABLE 7.—Effect
of feed additive antibiotics on mortality and gross lesions in chickens infected with E. tenella Average lesion score/number of bird died (days after infection)
Group Inf. Inf. Inf.
Inf. unmed. Uninf. unmed.
Expt. 6 Expt. 1
Expt. 2
Expt. 3
Expt. 4
Expt. 5
thiopeptin N.D. 1 N.D.
thiopeptin
thiopeptin
4.0/6 (5) N.D.
N.D. N.D.
bacitracin N.D. N.D.
penicillin N.D. 4.0/1 (5)
N.D.
N.D.
N.D.
4.0/1(7)
N.D.
N.D.
N.D. N.D.
N.D. N.D.
N.D. N.D.
N.D. N.D.
N.D. N.D.
N.D. N.D.
None
4.0/2 (5)
4.0/5 (5)
4.0/3 (5)
N.D.
4.0/1 (5)
4.0/3 (5)
None
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
antibiotic amprolium ethopabate antibiotic amprolium ethopabate clopidol antibiotic clopidol
chlortetracycline 4.0/2 (5) N.D.
'No. death. 1, severe cecal hemorrhage was observed in an infected, unmedicated group and infected fed dietary thiopeptin (Table 1). There were very few lesions (score of 1) in the ceca of one bird in infected medicated with amprolium plus ethopabate. Records on gross cecal lesions in dead birds are summarized in Table 7. Birds that died 5 days after infection were characterized by a presence of a large volume of blood in ceca and thickening of cecal wall (lesion score of 4). One male in Experiment 4 started to show bloody droppings 4 days after infection and died following 3 days of moribund conditions. Upon necropsy, cecal wall was distended and large caseous cores were found. DISCUSSION Under the conditions of these experiments, infected chickens on the basal ration and those on antibiotic diets were equally depressed by coccidia infection during the first 5 to 6 days after infection, and sometimes accompanied by deaths due to severe cecal haemorrhage. Nevertheless, infected birds responded to the antibiotic medications at the end of 3-week feedings and recovered from the depressed weights as indicated by
the percentages of relative weight. Similarly, when birds were infected with coccidiostatresistant E. tenella, supplementation of antibiotics and amprolium plus ethopabate in the diet resulted in higher percentages of relative weight than in amprolium plus ethopabate alone. On the contrary, in Experiment 3, clopidol and thiopeptin plus clopicol diets did not appear to bring advantageous effect on body weight as compared with other groups. This could possibly be related to the fact that the E. tenella used was clopidol-resistant strain (the anticoccidial index = 134 at 125 mg./kg.) but still slightly susceptible to the drug and, accordingly, relapse of infection due to a characteristic of clopidol's pharmaco-kinetics may have contributed to delayed depression of body weight, delayed recovery from disease syndrome, and delayed oocyst production. Studies in bacteria-free and gnotobiotic chickens have shown that C. perfringens and other bacteria are involved in producing the disease syndrome (Visco and Burns, 1972a, b; Bradley and Radhakrishnan, 1973). Previous studies demonstrated that the growth of C. perfringens stimulated by E. tenella infection in the ceca of chickens was suppressed by feeding thiopeptin, bacitracin,
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Inf. Inf.
Supplement in feed
1018
O. OHE AND A. ARAKAWA
REFERENCES
penicillin, or chlortetracycline (Arakawa and Ohe, 1975). In the present study, improvement of performance evaluated on the basis of body weight may in part be contributed by suppression of bacteria detrimental to host including C. perfringens. Gross pathologic lesions in the ceca of chickens withdrawn 7 days after
infection
in Experiment 1 agree with the results obtained in the previous study (Arakawa and in infected fed dietary thiopeptin in Experiments 1 and 3, 6 deaths that occurred in Experiment 2 seemed to be unrelated
to
this antibiotic. It is also conceivable that birds died in Experiments 4 and 6 are not attributed to feeding of bacitracin and chlortetracycline. Possible relationship between the use of antibiotics in chickens infected with coccidia and the clinical signs or mortality that might be associated with a change in microflora in intestine remains to be investigated. Results of the present study indicate that supplementation of antibiotics in the feed is advantageous to improve performance
of
chickens especially when chickens are on continuous threat by
coccidiostat-resistant
coccidia. ACKNOWLEDGEMENT The authors gratefully express their appreciation to Dr. K. Tsunoda of the National Institute of Animal Health, Tokyo, for sharing a strain of E. tenella; and to Miss Kita for her technical assistance.
NEWS AND NOTES (Continued from page 1007) II and Korea, currently holding the rank of Captain in the U.S. Naval Reserves. He has published 40 research reports and is coauthor of two books—"Sensory Deprivation," published by Harvard University Press, and "Psychological
Stress," published by Appleton, Century, Croft. He is also Director and Editor of the N.A.T.O. exercise and publication, "Environment Modification for Human Performance." Among the many special awards and honours he
(Continued on page 1030)
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Ohe, 1975). Since no bird died of coccidiosis
Arakawa, A., and O. Ohe, 1975. Reduction of Clostridium perfringens by feed additive antibiotics in the ceca of chickens infected with Eimeria tenella. Poultry Sci. 54: 1000-1007. Bradley, R. E., and C. V. Radhakrishnan, 1973. Coccidiosis in chickens: Obligate relationship between Eimeria tenella and certain species of cecal microflora in the pathogenesis of the disease. Avian Dis. 17: 461-476. Hein, H., and L. Timms, 1972. Bacterial flora in the alimentary tract of chickens infected with Eimeria brunetti and in chickens immunized with Eimeria maxima and cross-infected with Eimeria brunetti. Exp. Parasitol. 31: 188-193. Johansson, K. R., and W. B. Sarles, 1948. Bacterial population changes in the ceca of young chickens infected with Eimeria tenella. J. Bacterid. 56: 635648. Johnson, J., and W. M. Reid, 1970. Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments with chickens. Exp. Parasitol. 28: 30-36. Lafont, J. P., 1966. Flora intestinale et parasitoses: 1'exemple de la coccidiose caecale du poulet. Les cahiers de medicine veterinaire, 35: 257-280. McManus, E. C , W. C. Campbell and A. C. Cuckler, 1968. Development of resistance to quinoline coccidiostats under field and laboratory conditions. J. Parasitol. 54: 1190-1193. Steel, R. G. D., and J. H. Torrie. 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York, N.Y. Tsunoda, K , and T. Ishii, 1971. Niwatori no coccidium kensa ho. Keibyo Kenkyu Kai, 30 p. Visco, R. J., and W. C. Burns, 1972a. Eimeria tenella in bacteria-free and conventionalized chicks. J. Parasitol. 58: 323-331. Visco, R. J., and W. C. Burns, 1972b. Eimeria tenella in monoflora and diflora chicks. J. Parasitol. 58: 576-585.
Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 2-1-6, Kashima-cho, Osaka 532, Japan
Yodagawa-ku,
(Received for publication September 17, 1974)
POULTRY SCIENCE 54: 1008-1018, 1975
INTRODUCTION
S
EVERAL workers reported changes in the bacterial flora of intestinal tract of chickens as a result of coccidia infection. Johansson and Sarles (1948) reported increase of Clostridium perfringens and decrease of lactobacilli and enterococci in the ceca of chickens infected with Eimeria tenella. Similar findings were reported by Lafont (1966) and Bradley and Radhakrishnan (1973). Hein and Timms (1972) found significant increase of C. perfringens and Escherichia coli in small intestine and ceca of chickens infected with E. brunetti. Recent studies confirmed the increase of C. perfringens in the ceca of chickens infected with E. tenella and of coccidiostat-treated chickens infected with coccidiostat-resistant E. tenella, and showed that the increase was suppressed by supplementing antibiotics in the feed (Arakawa and Ohe, 1975). Antibiotics found to be effective
were thiopeptin at 2 mg./kg. of feed, bacitracin at 20 mg./kg., penicillin at 12 mg./kg., and chlortetracycline at 22 mg./kg. The purpose of the present experiments was to study what effect these antibiotics given via the feed would have on body weight, mortality and oocyst production in chickens infected with E. tenella and in coccidiostattreated chickens infected with coccidiostatresistant strains of E. tenella.
MATERIALS AND METHODS Birds. Day-old broiler chicks, Arbor AcreVantress crossbreed, were obtained from a commercial hatchery and were reared in conventional electrically heated battery brooders until use. They were caged in batteries with raised screen bottoms in a room of a heated building, ventilated and constructed to prevent excessive temperature changes.
1008
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ABSTRACT Six experiments were carried out to study the effect of feed additive antibiotics on body weight, mortality and oocyst production in chickens infected with coccidiostat-susceptible or -resistant strain of E. tenella. Levels of antibiotic and coccidiostat in fed (mg./kg.) were: thiopeptin, 2; zinc bacitracin, 20; penicillin, 12; chlortetracycline, 22; amprolium plus ethopabate, 125 plus 8; clopidol, 125. All experiments included 7 groups; 2 of these groups were uninfected and infected controls, and the 5 remaining groups were all infected and given diet containing antibiotic, amprolium plus ethopabate, antibiotic and amprolium plus ethopabate, clopidol, or antibiotic and clopidol. Chickens in each group were fed respective diet beginning one day prior to coccidia exposure. In two experiments, infection with a coccidiostat-susceptible strain resulted in severe clinical coccidiosis in chickens on the basal ration and on thiopeptin-diet, but dietary thiopeptin prompted recovery of body weight. In one experiment where chickens were infected with a strain resistant to amprolium plus ethopabate and clopidol, birds on dietary thiopeptin attained higher body weight than birds on the basal ration. In three experiments when a strain resistant to amprolium plus ethopabate was inoculated, birds given the basal ration, bacitracin, penicillin, chlortetracycline, or amprolium plus ethopabate diet developed cecal coccidiosis. Chickens on ration containing antibiotic alone attained higher body weight than chickens on the basal ration. Combination of antibiotic and amprolium plus ethopabate resulted in higher weight attained than amprolium plus ethopabate alone. Clopidol suppressed development of coccidiosis, and the combination of antibiotic and clopidol resulted in higher gains than in clopidol alone.
ANTIBIOTIC FEEDING IN COCCIDIOSIS
Diet. The basal ration used was reported in the previous study (Arakawa and Ohe, 1975). Medicated feed was prepared by mixing the basal ration with a measured amount of commercial premix. Levels of antibiotic and coccidiostat in feed (mg./kg.) were: thiopeptin, 1 2; zinc bacitracin, 2 20; procaine penicillin G, 3 12; chlortetracycline hydrochloride, 4 22; amprolium plus ethopabate, 5 125 plus 8; and clopidol, 6 125.
Coccidial Challenge. Coccidiostat-susceptible strain of E. tenella was maintained by Dr. K. Tsunoda of the National Institute of Animal Health in Tokyo and supplied to this laboratory. This strain was used in Experiments 1 and 2. Two coccidiostat-resistant strains of E. tenella were obtained from litter
1. 2. 3. 4. 5. 6.
Fujisawa Pharmaceutical Co., Ltd., Osaka. Nippon Kayaku Co., Ltd., Osaka. Taito Pfizer Co., Ltd., Tokyo. Takeda Chemical Industries, Osaka. Marupi-Merck Sharp and Dohme, Osaka. Tanabe Co., Ltd., Osaka.
samples from local broiler houses, separated by a single oocyst method, and propagated through chickens under strictly isolated conditions. These strains were tested previously by the methods described by McManus et al. (1968). One strain used in Experiment 3 was resistant to amprolium plus ethopabate (the anticoccidial index = 130 at 125 plus 8 mg./kg. in the feed) and clopidol (the index = 134 at 125 mg./kg.), and another strain used in Experiments 4, 5, and 6 was resistant to amprolium plus ethopabate (the index = 121 at 125 plus 8 mg./kg.). Fresh oocyst cultures were prepared routinely from donor chickens 7 to 8 days after infection. Chickens in infected groups were each given an oral inoculation of 30,000 sporulated oocysts. Daily oocyst counts were made starting 7 (Experiments 3,4,5, and6)or 8 (Experiment 1) days after infection. Fecal material was collected from each cage and was well mixed to obtain estimates of oocyst production. Appropriate aliquots were taken, diluted, and placed on a plankton counting plate (Tsunoda and Ishii, 1971). Birds that were withdrawn during the Experiment 1 and those that died were necropsied for gross pathological observations. Cecal lesions were scored using a 0 to 4 system as described by Johnson and Reid (1970). Statistical Methods. Analysis of variance and Duncan's new multiple range test (P = 0.05) were performed for statistical evaluation of differences in body weight among 7 groups (Steel and Torrie, 1960). Experiment 1. Seventeen-day-old chickens, males weighing 280 to 285 g. and females, 270 to 275 g., were used. Rations containing thiopeptin and coccidiostats were fed consecutively for 14 days. Infected groups were exposed to coccidiostat-susceptible strain of E. tenella. Seven days after infection 10 birds, 5 males and 5 females, were randomly selected from each group and necropsied for macro-
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Effect of Antibiotics on Chickens. Six experiments were conducted. In each experiment, a total of 140 chickens were divided into uniform groups of 7 consisting of 10 males and 10 females each on the basis of body weight. The groups consisted of those infected fed a ration containing either antibiotic, amprolium plus ethopabate, antibiotic and amprolium plus ethopabate, clopidol, or antibiotic and clopidol. The two controls were an infected, unmedicated group and an uninfected, unmedieated group. The birds in each group received respective ration beginning one day before coccidia exposure and were fed continuously until the termination of experiments. Water was available ad libitum. Birds were individually weighed daily during the first 6 or 7 days and weekly thereafter, excepting Experiment 6 that terminated 18 days after infection.
1009
416
485 ab 515 a
479 abc
469 ab 467 ab
513 a
None
455 a b c
494 ab
421 b c
421 b c 475 a
450 "°
2 125 8 2 125 8 125 2 125
Female
None
thiopeptin amprolium ethopabate thiopeptin amprolium ethopabate clopidol thiopeptin clopidol
Male 432=
6 days (10 birds) abc
7)8
abc
705 bc
675 c 808 a
778 a
661 c
722
Male 675 ab 665 ab
97.3 100
596 b
106.5 109.7
658 ab
671 a b 693 a
651 a b 108.1
95.3 108.2
Female
Yo
relativ weight 6 days
13 days (5 birds)
Average live weight (g.)'
'Within each column, any averages having the same superscript are not significantly different at 0.05 2 Days after coccidia infection. 'Average of 8 birds (5 males and 3 females).
Inf. unmed. Uninf. unmed.
Inf. Inf.
Inf.
Inf. Inf.
Group
2
of thiopeptin and coccidiostats on chickens infected with a coccidiostat-suscep
Supplement in feed (mg./kg.)
TABLE 1.—Effect
om http://ps.oxfordjournals.org/ at California Institute of Technology on June 20, 2015
1011
ANTIBIOTIC FEEDING IN COCCIDIOSIS
scopical observations with the exception of infected control where only 8 birds (5 males and 3 females) were necropsied. Daily oocyst counts were made in the 5 remaining birds.
Experiment 3. Birds, 14 days old, were used in 21-day experiment. Their weight ranged from 200 to 220 g. for males and 190 to 210 g. for females. Feed supplemented with thiopeptin and coccidiostats was given. Chickens in infected groups were each given an oral inoculation of E. tenella oocysts that are resistant to amprolium plus ethopabate and clopidol.
Experiment 5. The average body weight of the birds on the 21-day feeding trial were males 245 g. and females 235 g. The same strain of E. tenella was used to infect the infected group as in Experiment 4. Experiment 6. Eighteen-day-old chickens, weighing an average of 330 g. for males and 325 g. for females, were used for 19-day study. Feed was medicated with chlortetracycline and coccidiostats. Coccidia inoculated were the same strain used in Experiment 4. RESULTS Experiment 1. Table 1 summarizes the results. Females in uninfected control group were attracted to the feed much slower than
TABLE 2.- -Effect of thiopeptin and coccidiostats on chickens infected with a coccidiostat-susceptible
strain ofE. tenella (Experiment 2)
% re lative w eight
Average live weight (g.)' 6 days 2 Group Inf. Inf. Inf.
Inf. Inf. Inf. unmed. Uninf. unmed.
Feed (mg./kg.) thiopeptin amprolium ethopabate thiopeptin amprolium ethopabate clopidol thiopeptin clopidol
2 125 8 2 125 8 125 2 125
Male 150 194a
Female 132 175b
20 days Male
6 days
20 days
545 606 ab
560a 567"
76.6 100.2
98.1 104.4
Female
189ab
195
575 c
572 a
104.3
101.8
194a 195a
183 ab 175b
628 a 589 >*
548 ab 534"
102.4 100.5
104.4 99.7
None
171
141
593 ^
486 c
84.7
95.8
None
184"
184a
623 a
503 c
100
100
'Within each column, any averages having the same superscript are not significantly different at 0.05 level of probability. 2 Days after coccidia infection.
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Experiment 2. Chickens, 7 days old, ranging from 70 to 95 g. of body weight in both sexes were used. This experiment was designed to observe change in body weight for longer period than in Experiment 1; no chickens were withdrawn except birds that died. Feed containing thiopeptin and coccidiostats was given continuously for 21 days. Coccidia inoculated were the same strain used in Experiment 1.
Experiment 4. Chickens, 14 days old, were used. Feed containing bacitracin and coccidiostats was given for 21 days. Birds in the infected groups were each exposed to E. tenella that are resistant to amprolium plus ethopabate.
thiopeptin amprolium ethopabate thiopeptin amprolium ethopabate clopidol thiopeptin clopidol
2 125 8 2 125 8 125 2 125 791 a
801 a 765 a
919 a
869 a 869 a
395 a 366 bc
4 0 7 ab 403 ab
796 a 790 a
353 cd
388"
348 c
387 b
20 days Female
Male 882 a 890 a
Female 326= 3 5 5 cd
6 days
Male
%
98.1 94.2
90.5
relative weight 6 days 20 82.5 90.9
Inf. 764 a None 345 c 876 a 328 d unmed. 82.3 Uninf. 429 a 924 a 841 a None unmed. 100 1 388 a 'Within each column, any averages having the same superscript are not significantly different at 0.05 2 Days after coccidia infection.
Inf. Inf.
Inf.
Inf. Inf.
Group
2
Average live weight (g.)'
of thiopeptin and coccidiostats on chickens infected with a strain o / E . tenella resistan (Experiment 3)
Supplement in feed (mg./kg.)
TABLE 3.—Effect
m http://ps.oxfordjournals.org/ at California Institute of Technology on June 20, 2015
ANTIBIOTIC FEEDING IN COCCIDIOSIS
the birds in other groups. This was reflected in their lower weight on 6 and 13 days after infection. Average weights on 6 days after infection of birds given the basal ration and dietary thiopeptin were significantly (P < 0.05) lower than those receiving coccidiostats. Oocyst discharge in feces was not affected by thiopeptin medication.
Experiment 3. Infection with coccidiostatresistant E. tenella resulted in weight depression in all birds on 6 days after infection (Table 3). Infected chickens receiving the basal feed and dietary thiopeptin were significantly (P < 0.05) lower than the rest of groups. Average weight of chickens given amprolium plus ethopabate was significantly lower (P < 0.05) than those in uninfected control. Birds on dietary clopidol were behind in their weight as compared with uninfected control, but the difference was not statistically significant (P > 0.05). Weights of all infected groups taken on 20 days after infection were lower than those of uninfected control, but there were no statistical differences (P > 0.05) among 7 groups. Infected chickens on medicated diets discharged oocysts comparable to those on the basal ration, but the birds given clopidol diet showed delayed and prolonged discharge of oocysts. Experiment 4. Table 4 contains the results. There were no statistical differences (P > 0.05) in weight on 5 and 20 days after infection
between uninfected group and bacitracintreated infected group. Clopidol prevented an outbreak of coccidiosis, and a combination with bacitracin resulted in better performance than in clopidol alone. Percentages of relative weight of birds on dietary bacitracin were greater than those on the diet without bacitracin. Oocyst counts in infected birds given amprolium plus ethopabate diet were fewer than those in infected control. Experiment 5. Results are presented in Table 5. Body weight of birds in infected control group was the lowest (P < 0.05) among 7 groups on 5 and 20 days after infection. Medication of amprolium plus ethopabate alone did not fully protect from coccidia infection as evaluated by body weight on 5 days after infection and by oocyst production. Feeding the ration containing penicillin and amprolium plus ethopabate resulted in better performance than amprolium plus ethopabate alone. Oocyst discharge was not influenced by penicillin medication. Experiment 6. Results are shown in Table 6. Weights on 5 days after infection of infected birds on the basal ration, on chlortetracycline diet, and amprolium plus ethopabate diet were significantly (P < 0.05) lower than those in uninfected control group. By 18 days after infection, the birds on chlortetracycline diet recovered their weight equal to uninfected control (P > 0.05), whereas infected birds on the basal feed and those on amprolium plus ethopabate diet did not recover. Chickens on feed supplemented with either chlortetracycline alone or combined with the coccidiostats attained greater body weight on 18 days after infection than those given without antibiotic. Clopidol suppressed coccidiosis. Oocyst discharge was not affected by chlortetracycline medication. Gross Pathology. In birds that were withdrawn 7 days after infection in Experiment
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Experiment 2. Body weight on 6 days after infection of the surviving birds given the basal feed and dietary thiopeptin was significantly (P < 0.05) lower than those of the rest of groups (Table 2). Percentage of relative weight indicated that birds fed dietary thiopeptin lost more weight by 6 days after infection than those in infected control, but by 20 days after infection the birds on thiopeptin-diet attained greater body weight than the infected control.
1013
851 a 841 a b
358 ^
None
876 d 939 a
325 b 345 a
347" 344a
892 cd 1,010
342 375 a
835 a b
911bc
332 b
356 ^
845 a
821 b c
845 a 802 c
Female
920 ab 930 a
352" 331 b
20 days
366 a b 365 a b
353 c
20 125 8 20 125 8 125 20 125
None
bacitracin amprolium ethopabate bacitracin amprolium ethopabate clopidol bacitracin clopidol
Male
Female
5 days
Male
100
96.4
98.0 102.2
97.8
102.1 99.0
% relative weight 20 5 days
'Within each column, any averages having the same superscript are not significantly different at 0.05 2 Days after coccidia infection.
Inf. unmed. Uninf. unmed.
Inf. Inf.
Inf.
Inf. Inf.
Group
2
Average live weight (g.)'
of bacitracin and coccidiostats on chickens infected with a strain of E. tenella r (Experiment 4)
Supplement in feed (mg./kg.)
TABLE 4.—Effect
m http://ps.oxfordjournals.org/ at California Institute of Technology on June 20, 2015
878 c
394 a
41 5 bc
862 c 960 a
None
387 a 394"
413 bc 431 a
927 b
812
385 a
423 ab
925" 953"
358
383 a 373
412 c 408 c
386
12 125 8 12 125 8 125 12 125
Male
None
penicillin amprolium ethopabate penicillin amprolium ethopabate clopidol penicillin clopidol
Female
5 days
Male
91.9 100
820 bc
98.8 102.0
100
98.2 96.5
5 days
758
833 ab 831 a b
850"
800 c
817bc
Female
20 days
%
relati weig
Within each column, any averages having the same superscript are not significantly different at 0.0 2 Days after coccidia infection.
1
Inf. unmed. Uninf. unmed.
Inf. Inf.
Inf.
Group Inf. Inf.
2
Average live weight (g.)'
of penicillin and coccidiostats on chickens infected with a strain ofE. tenella resista
Supplement in feed (mg./kg.)
TABLE 5.—Effect
m http://ps.oxfordjournals.org/ at California Institute of Technology on June 20, 2015
499"
492 be 516 a
534 b
549 a 560 a c
953 c 982 b
l,040 bc l,060 ab
522 a
535"
None
l,010 a
939
992 ab
Female 99 0 ab 922
475 d
1,096 1,121
l,069 a
l,038 b c 1,023c
487 c 475 d
513 c 512°
509 c
22 125 8 22 125 8 125 22 125
18 days
None
chlortetracycline amprolium ethopabate chlortetracycline amprolium ethopabate clopidol chlortetracycline clopidol
Male
Female
5 days
Male
100
93.1
98.4 101.8
97.7
94.6 93.3
5 days
% relativ weigh
'Within each column, any averages having the same superscript are not significantly different at 0.0 2 Days after coccidia infection.
Inf. unmed. Uninf. unmed.
Inf. Inf.
Inf.
Inf. Inf.
Group
2
Average live weight (g.)'
of chlortetracycline and coccidiostats on chickens infected with a strain of E. ten (Experiment 6)
Supplement in feed (mg./kg.)
TABLE 6.—Effect
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1017
ANTIBIOTIC FEEDING IN COCCIDIOSIS
TABLE 7.—Effect
of feed additive antibiotics on mortality and gross lesions in chickens infected with E. tenella Average lesion score/number of bird died (days after infection)
Group Inf. Inf. Inf.
Inf. unmed. Uninf. unmed.
Expt. 6 Expt. 1
Expt. 2
Expt. 3
Expt. 4
Expt. 5
thiopeptin N.D. 1 N.D.
thiopeptin
thiopeptin
4.0/6 (5) N.D.
N.D. N.D.
bacitracin N.D. N.D.
penicillin N.D. 4.0/1 (5)
N.D.
N.D.
N.D.
4.0/1(7)
N.D.
N.D.
N.D. N.D.
N.D. N.D.
N.D. N.D.
N.D. N.D.
N.D. N.D.
N.D. N.D.
None
4.0/2 (5)
4.0/5 (5)
4.0/3 (5)
N.D.
4.0/1 (5)
4.0/3 (5)
None
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
antibiotic amprolium ethopabate antibiotic amprolium ethopabate clopidol antibiotic clopidol
chlortetracycline 4.0/2 (5) N.D.
'No. death. 1, severe cecal hemorrhage was observed in an infected, unmedicated group and infected fed dietary thiopeptin (Table 1). There were very few lesions (score of 1) in the ceca of one bird in infected medicated with amprolium plus ethopabate. Records on gross cecal lesions in dead birds are summarized in Table 7. Birds that died 5 days after infection were characterized by a presence of a large volume of blood in ceca and thickening of cecal wall (lesion score of 4). One male in Experiment 4 started to show bloody droppings 4 days after infection and died following 3 days of moribund conditions. Upon necropsy, cecal wall was distended and large caseous cores were found. DISCUSSION Under the conditions of these experiments, infected chickens on the basal ration and those on antibiotic diets were equally depressed by coccidia infection during the first 5 to 6 days after infection, and sometimes accompanied by deaths due to severe cecal haemorrhage. Nevertheless, infected birds responded to the antibiotic medications at the end of 3-week feedings and recovered from the depressed weights as indicated by
the percentages of relative weight. Similarly, when birds were infected with coccidiostatresistant E. tenella, supplementation of antibiotics and amprolium plus ethopabate in the diet resulted in higher percentages of relative weight than in amprolium plus ethopabate alone. On the contrary, in Experiment 3, clopidol and thiopeptin plus clopicol diets did not appear to bring advantageous effect on body weight as compared with other groups. This could possibly be related to the fact that the E. tenella used was clopidol-resistant strain (the anticoccidial index = 134 at 125 mg./kg.) but still slightly susceptible to the drug and, accordingly, relapse of infection due to a characteristic of clopidol's pharmaco-kinetics may have contributed to delayed depression of body weight, delayed recovery from disease syndrome, and delayed oocyst production. Studies in bacteria-free and gnotobiotic chickens have shown that C. perfringens and other bacteria are involved in producing the disease syndrome (Visco and Burns, 1972a, b; Bradley and Radhakrishnan, 1973). Previous studies demonstrated that the growth of C. perfringens stimulated by E. tenella infection in the ceca of chickens was suppressed by feeding thiopeptin, bacitracin,
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Inf. Inf.
Supplement in feed
1018
O. OHE AND A. ARAKAWA
REFERENCES
penicillin, or chlortetracycline (Arakawa and Ohe, 1975). In the present study, improvement of performance evaluated on the basis of body weight may in part be contributed by suppression of bacteria detrimental to host including C. perfringens. Gross pathologic lesions in the ceca of chickens withdrawn 7 days after
infection
in Experiment 1 agree with the results obtained in the previous study (Arakawa and in infected fed dietary thiopeptin in Experiments 1 and 3, 6 deaths that occurred in Experiment 2 seemed to be unrelated
to
this antibiotic. It is also conceivable that birds died in Experiments 4 and 6 are not attributed to feeding of bacitracin and chlortetracycline. Possible relationship between the use of antibiotics in chickens infected with coccidia and the clinical signs or mortality that might be associated with a change in microflora in intestine remains to be investigated. Results of the present study indicate that supplementation of antibiotics in the feed is advantageous to improve performance
of
chickens especially when chickens are on continuous threat by
coccidiostat-resistant
coccidia. ACKNOWLEDGEMENT The authors gratefully express their appreciation to Dr. K. Tsunoda of the National Institute of Animal Health, Tokyo, for sharing a strain of E. tenella; and to Miss Kita for her technical assistance.
NEWS AND NOTES (Continued from page 1007) II and Korea, currently holding the rank of Captain in the U.S. Naval Reserves. He has published 40 research reports and is coauthor of two books—"Sensory Deprivation," published by Harvard University Press, and "Psychological
Stress," published by Appleton, Century, Croft. He is also Director and Editor of the N.A.T.O. exercise and publication, "Environment Modification for Human Performance." Among the many special awards and honours he
(Continued on page 1030)
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Ohe, 1975). Since no bird died of coccidiosis
Arakawa, A., and O. Ohe, 1975. Reduction of Clostridium perfringens by feed additive antibiotics in the ceca of chickens infected with Eimeria tenella. Poultry Sci. 54: 1000-1007. Bradley, R. E., and C. V. Radhakrishnan, 1973. Coccidiosis in chickens: Obligate relationship between Eimeria tenella and certain species of cecal microflora in the pathogenesis of the disease. Avian Dis. 17: 461-476. Hein, H., and L. Timms, 1972. Bacterial flora in the alimentary tract of chickens infected with Eimeria brunetti and in chickens immunized with Eimeria maxima and cross-infected with Eimeria brunetti. Exp. Parasitol. 31: 188-193. Johansson, K. R., and W. B. Sarles, 1948. Bacterial population changes in the ceca of young chickens infected with Eimeria tenella. J. Bacterid. 56: 635648. Johnson, J., and W. M. Reid, 1970. Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments with chickens. Exp. Parasitol. 28: 30-36. Lafont, J. P., 1966. Flora intestinale et parasitoses: 1'exemple de la coccidiose caecale du poulet. Les cahiers de medicine veterinaire, 35: 257-280. McManus, E. C , W. C. Campbell and A. C. Cuckler, 1968. Development of resistance to quinoline coccidiostats under field and laboratory conditions. J. Parasitol. 54: 1190-1193. Steel, R. G. D., and J. H. Torrie. 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York, N.Y. Tsunoda, K , and T. Ishii, 1971. Niwatori no coccidium kensa ho. Keibyo Kenkyu Kai, 30 p. Visco, R. J., and W. C. Burns, 1972a. Eimeria tenella in bacteria-free and conventionalized chicks. J. Parasitol. 58: 323-331. Visco, R. J., and W. C. Burns, 1972b. Eimeria tenella in monoflora and diflora chicks. J. Parasitol. 58: 576-585.
