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Reduction of Clostridium perfringens by Feed Additive Antibiotics in the Ceca of Chickens Infected with Eimeria tenella
1000
W. A. BOUGH, A. L. SHEWFELT AND W. L. SALTER
Mercer, W. A., 1969. A guide for waste management in the food processing industries. National Canners Assoc, Berkeley, Calif. Peniston, Q. P., and E. L. Johnson, 1970. Method for treating an aqueous medium with chitosan and derivatives of chitin to remove an impurity. U.S. Patent No. 3,533,940. Perceval, P. M., 1974. Personal communication, December 18. Singh, S. P., R. L. Wesley and E. A. Budd, 1973. Characteristics of poultry processing effluent. Poultry Sci. 52: 1478-1481. Whitehead, W. K., 1974. Analysis of some physical properties of poultry processing chiller effluent. Poultry Sci. 53: 571-573.
Reduction of Clostridium perfringens by Feed Additive Antibiotics in the Ceca of Chickens Infected with Eimeria tenella A. ARAKAWA AND O. OHE
Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 2-1-6, Kasima-cho, Yodogawa-ku, Osaka 532, Japan (Received for publication September 17, 1974)
ABSTRACT Two experiments were performed to investigate the effect of feed additive antibiotics on Clostridium perfringens and Enterobacteriaceae in the ceca of chickens infected with Eimeria tenella. In the first experiment, chickens were continuously fed rations containing thiopeptin, 2 mg./kg.; bacitracin, 20 mg./kg.; penicillin, 12 mg./kg.; or chlortetracycline, 22 mg./kg. One day after antibiotic feed was given, each bird received an oral inoculation of 30,000 E. tenella oocysts. The growth of C. perfringens was stimulated by the infection in unmedicated chickens. Dietary thiopeptin, bacitracin, penicillin, or chlortetracycline suppressed the number of C. perfringens recovered 5 and 7 days after infection. Enterobacteriaceae were increased by the infection, but dietary antibiotics did not reduce the increase. In the second experiment, chickens were given feed containing amprolium plus ethopabate, 125 plus 8 mg./kg., and a combination of the coccidiostat and one of 4 antibiotics: thiopeptin, bacitracin, penicillin, or chlortetracycline. Birds were each given an oral inoculation of 30,000 coccidiostat-resistant E. tenella oocysts. Infection resulted in an increase of C. perfringens in the unmedicated control and the coccidiostat-treated groups. Dietary thiopeptin, bacitracin, penicillin, or chlortetracycline reduced the number of C. perfringens found 5 and 7 days after infection. Counts of Enterobacteriaceae were increased by the infection, but dietary antibiotics did not suppress the increased counts. In both experiments, dietary administration of antibiotics did not reduce gross cecal lesions. POULTRY SCIENCE 54: 1000-1007, 1975
INTRODUCTION
C
HANGES in the bacterial flora of the
intestinal tract of chickens infected with
enterococci in the ceca of chickens infected with Eimeria tenella. Similar findings were observed by Lafont (1966) and Bradley and
coccidia have been reported. Johansson and
Radhakrishnan
Sarles (1948) found an increase of Clostridium
(1972) reported a significant increase of Es-
(1973).
Hein
and
Timms
perfringens and decrease of lactobacilli and
cherichia coli and C. perfringens in the small
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CRESA (Food, Chemical and Research Laboratories, Inc. and Engineering Science of Alaska), 1971. Pollution abatement and by-product recovery in shellfish and fisheries processing. Environmental Protection Agency, Project No. 12130FJQ, Supt. of Documents, Washington, D.C. Culp, R. L., and G. L. Culp, 1971. Advanced Wastewater Treatment. VanNostrand Reinhold Company, New York, N.Y., p. 256. Hamm, D., 1972. Characteristics of effluents in ten Southeastern poultry processing plants. Poultry Sci. 51:825-829. Mauldin, A. F., 1974. Case study—treatment of Gulf shrimp processing and canning wastes. Environmental Protection Agency, Technology Transfer Seminar, New Orleans, La., March 5-6.
ANTIBIOTICS AND CLOSTRIDIUM PERFRINGENS
TABLE 1.—Composition of basal ration Ingredients % Ground yellow corn 51.40 Soybean meal 20.00 Ground milo 14.00 Fish meal 8.00 Alfalfa meal 3.00 Calcium carbonate 1.50 Tricalcium phosphate 1.00 Salt 0.50 DL-methionine 0.20 Micronutrients' 0.40 1 Micronutrients provided in mg. and in units per kg. of ration: thiamine, 2 mg.; riboflavin, 10 mg.; pyridoxine, 1 mg.; niacin, 6 mg.; calcium pantothenate, 6.5 mg.; choline chloride, 120 mg.; folic acid, 0.2 mg.; vitamin A, 10,000 U.; vitamin D 3 , 2,000 U.; vitamin E, 10 mg.; manganese, 166 mg.; iron, 60 mg.; copper, 6 mg.; cobalt, 0.2 mg.; zinc, 30 mg.
MATERIALS AND METHODS Birds. White Leghorn, Hy-Line®, cockerels were obtained from a local commercial hatchery when less than one day old. They were reared in conventional electrically heated battery brooders and fed the basal ration until use. They were caged in batteries in air-conditioned rooms with continuous artificial illumination. Diets. The composition of the basal ration is given in Table 1. Medicated feeds were prepared by mixing the basal ration with a measured amount of commerical 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; and amprolium plus ethopabate, 5 125 plus 8. Experiment 1. A total of 60 11-day-old birds, averaging 97 g. of body weight, were divided into 6 groups of 10 chickens each. The groups consisted of those infected fed a ration containing either thiopeptin, bacitracin, penicillin, or chlortetracycline. The two groups were an infected, unmedicated group and an uninfected, unmedicated group. The respective rations and water were given ad libitum beginning one day prior to coccidia exposure until 7 days after infection. A strain of E. tenella used was maintained by Dr. K. Tsunoda of the National Institute of Animal Health in Tokyo and supplied to this laboratory. Fresh oocyst cultures were prepared routinely from donor chickens 7 to 8 days after infection. Oocysts were washed with sterile saline solution six times immediately before use and 0.1 ml. of the suspension was
1. 2. 3. 4. 5.
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.
Downloaded from http://ps.oxfordjournals.org/ at University of Arizona on June 7, 2016
intestine and ceca of chickens infected with E. brunetti. In gnotobiotic chickens, Visco and Burns (1972b) and Bradley and Radhakrishnan (1973) demonstrated that not only C. perfringens but also other bacteria including E. coli were responsible in part for the development of cecal coccidiosis. Recent studies in bacteria-free and conventional chicks have shown that germ-free chicks were less susceptible than conventionals to cecal coccidiosis (Visco and Burns, 1972a; Radhakrishnan and Bradley, 1973) and that gross lesions in germ-free chickens were less severe than those found in conventional infected chickens (Johnson et al., 1973). These findings and prevalent use of antibiotics in the current poultry feed stimulated the investigators to determine if antibiotics given via the feed would suppress the increase of C. perfringens stimulated by E. tenella infection and influence gross lesions. At the same time, a similar investigation was directed toward Enterobacteriaceae. Two studies were made: E. tenella infection in the presence of antibiotics in the feed and coccidiostat-resistant E. tenella infection in the presence of antibiotics and coccidiostat.
1001
1002
A. ARAKAWA AND O. OHE
Experiment 2. A total of 70 chickens, 11 days old, were allotted into 7 groups of 10 birds each. The groups consisted of those infected fed a ration containing amprolium plus ethopabate, and a combination of the coccidiostat and one of 4 antibiotics, i.e., thiopeptin, bacitracin, penicillin, or chlortetracycline. The two groups were an infected, unmedicated group and an uninfected, unmedicated group. The respective rations and water were available ad libitum starting one day before coccidia exposure until 7 days after infection. The E. tenella used was a coccidiostat-resistant strain which was obtained from litter samples from a local broiler house, separated by a single-oocyst method, and propagated through chickens under strictly isolated conditions. This strain was tested previously by the methods described by McManus et al. (1968) and found to be resistant to amprolium plus ethopabate, 125 plus 8 mg./kg., in the feed (the anticoccidial index = 121). Oocysts were washed with sterile saline six times before exposure and tested for sterility. Chickens in infected groups each received a single oral dose of 30,000 sporulated oocysts. Necropsy procedures were the same as those in the previous experiment. Bacteriological
Examination,
CW agar (5
g. of heart muscle infusion (Nissan), 10 g. of proteose peptone W (Nissan), 10 g. of casein peptone (Nissan), 5 g. of sodium chloride, 10 g. of lactose, 0.05 g. of phenol red, 20 g. of agar, and 0.2 g. of kanamycin sulfate per 1,000 ml. of distilled water; Nissui Kagaku Co., Ltd., Tokyo) was used to recover C. perfringens. DHL agar (Eiken Kagaku Co., Ltd., Tokyo) was used to count a total population of Enterobacteriaceae (Mitsuoka et al., 1965). EMB agar was utilized to enumerate E. coli and Aerobacter spp. Oocyst suspensions used for infecting chickens were tested for sterility by spreading 0.1 ml. over CW and DHL plates. At necropsy, ceca were isolated and midportion was opened under aseptic procedures. Cecal samples from a single animal were placed in a weighed and sterilized anaerobic glass tube containing approximately 120 glass beads (2.5 mm. in diameter). Tubes were weighed and the initial dilution was made by adding 9 volumes of sterile anaerobic diluent (4.5 g. of K H 2 P 0 4 , 6.0 g. of N a 2 H P 0 4 , 0.5 g. of L-cysteine-HCl • H 2 0 , 0.5 g. of Tween 80, 1.0 g. of agar and 1,000 ml. of distilled water). The tube was shaken until a homogenized suspension was obtained. One ml. of the suspension was withdrawn and further diluted in sterile anaerobic diluent by serial 10-fold steps. From each of the serial dilutions including the initial dilution, 0.1 ml. of suspension was taken, placed and spread on CW agar. Plates were incubated anaerobically in a Gaspak jar (with disposable hydrogen plus carbon dioxide generator envelopes, BBL) at 37° C. for 48 hours. Colonies characterized by lecithinase production were counted. A 0.1 ml. of suspension from each of the serial dilutions was spread over DHL and EMB agar plates. They were incubated aerobically at 37° C. for 24 hours. Colonies of E. coli characterized by a convex surface of 3 to 4 mm. in diameter appearing blackmetallic gold were counted. For identification, TSI agar, SIM medium, and Voges-
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provided for bacteriological examination. Birds in infected groups were each given an oral inoculation of 30, 000 sporulated oocysts. Five chickens in each group were killed 5 days after infection and the remaining animals were killed 2 days later for gross observation of the ceca and the bacteriological examination. At necropsy, cecal lesion scores were graded as described by Johnson and Reid (1970). They used a 0 to 4 scoring system with 1 for very few lesions, 2 for slight lesions, 3 for many lesions and considerable blood, and 4 for severe lesions and large amount of blood.
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Experiment 2. Table 3 contains the results. The counts of C. perfringens were significantly increased by a coccidiostat-resistant E. tenella infection in infected controls and
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Experiment I. Results are presented in Table 2. The increased number of C. perfringens detected 5 and 7 days after E. tenella infection in non-antibiotic treated controls was suppressed significantly by dietary administration of thiopeptin, bacitracin, penicillin, or chlortetracycline (P < 0.05). Enterobacteriaceae were increased by the coccidia infection; the increase was statistically significant only 7 days after infection (P < 0.05), but dietary antibiotics did not suppress the increase (P > 0.05). The growth of E. coli was similarly stimulated by the infection 5 and 7 days after infection, but the growth was statistically significant only 7 days after infection (P < 0.05). However, the growth was not suppressed by antibiotics in the diet (P > 0.05). There were no statistical differences in counts of Aerobacter spp. among 6 groups of chickens killed 5 and 7 days after infection (P > 0.05). No colonies were found on CW and DHL agar plates that were streaked with the oocyst suspension used for the infection.
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Statistical Analysis. Data on number of bacteria after square root transformation and cecal lesion scores were submitted to analysis of variance and Duncan's new multiple range test (P = 0.05) (Steel and Torrie, 1960).
D.
5 days-
Proskauer broth were used (Rohde, 1970). Colonies of Aerobacter spp. were counted. They were characterized by a concave surface of 4 to 6 mm. in diameter with brown centers. When no colony was found on agar plates with the initial dilution of suspension, the count was expressed as less than 100 per g. of cecal contents.
PERFRINGENS
ays
ANTIBIOTICS AND CLOSTRIDIUM
amprolium ethopabate thiopeptin amprolium ethopabate bacitracin amprolium ethopabate penicillin amprolium ethopabate chlortetracycline amprolium ethopabate
125 8 2 125 8 20 125 8 12 125 8 22 125 8 <10 2 a
<102
<10 2 a
<10 2 a
<10 2 a
<10 2 a
<10 2 a
OO23
2.7 x 108a
4.0 x 108a
1.9 x 10 8ab
6.3 x 107bc
2.9 x 109ab
2.5 x 109ab
4.4 x 109ab
2.7 x 109ab
1.0 x 10
1.4 x 10
2.5 x 10
2.4 x 10
Average number of organisms/g. of c Enterobacteriaceae 5 days 7 days 5 day 4.0 x 108a 1.8 x 109b 8.4 x 10
Inf. ;.6 x 106 3.6 x 108a 8.6 x 109a 4.0 x 10 unmed. None 1.2 x 103 Uninf. None unmed. <10 2 a <10 2 a 2.1 x 107c 5.2 x 107 7.0 x 10 '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.
Group Inf.
C. perfringens 5 days 7 days 3.4 x 104 :.2 x 107
of feed additive antibiotics on C. perfringens and Enterobacteriaceae in the ceca of strain of E. tenella (Experiment 2)
Supplement in feed (mg./kg.)
TABLE 3.—Effect
ded from http://ps.oxfordjournals.org/ at University of Arizona on June 7, 2016
ANTIBIOTICS AND CLOSTRIDIUM PERFRINGENS
in infected and amprolium plus ethopabate treated birds (P < 0.05). Dietary antibiotics combined with the coccidiostat prevented the increase of C. perfringens (P < 0.05). A total Enterobacteriaceae population was increased by the coccidia infection in non-antibiotic treated controls and in amprolium plus ethopabate medicated birds 5 and 7 days after exposure (P < 0.05). This increase was suppressed by dietary thiopeptin only 5 days after infection. E. coli was increased by coccidia infection, but significant increase was noted
1005
only 7 days after infection (P < 0.05). Dietary thiopeptin and bacitracin reduced the counts 5 days after infection and chlortetracycline suppressed E. coli 7 days after infection. Aerobacter spp. were significantly increased by coccidia infection (P < 0.05). Dietary thiopeptin reduced the increase only 5 days after infection. No colonies were formed on CW and DHL agar plates streaked with the oocyst inocula used.
Inf.
Inf.
Inf. Inf. unmed. Uninf. unmed.
amprolium ethopabate bacitracin amprolium ethopabate penicillin amprolium ethopabate chlortetracycline amprolium ethopabate
125 8 20 125 8 12 125 8 22 125 8
4.0 a
4.0 a
4.0 a
4.0 a
4.0 a
4.0 a
None
4.0 a
4.0 a
None
0
0
'Within each column, any averages having the same superscript are not significantly different at 0.05 level of probability. 2 Days after coccidia infection.
DISCUSSION The present study confirms the findings that the growth of C. perfringens in the ceca was stimulated by E. tenella infection (Johansson and Sarles, 1948; Bradley and Radhakrishnan, 1973) and demonstrates that the growth was equally stimulated by a coccidiostat-resistant E. tenella infection in the presence of coccidiostat in the feed. Johansson and Sarles (1948) found C. perfringens -like colonies up to 10" 8 dilution on the 5th day of infection although no counts were reported on the 7th day, whereas the counts in the
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Gross Pathology. Table 4 summarizes gross cecal lesion scores obtained 5 and 7 days after infection. In Experiment 1, a sublethal TABLE 4.—Effect of feed additive antibiotics on dose of coccidia resulted in severe lesions gross lesions of the ceca of chickens infected with with large volume of blood in ceca of all E. tenella (Experiments 1 and 2) birds in infected groups. Lesion scores among Supplement these groups were not significantly different i \ \ . lesion score' in feed (P > 0.05). There was no pathological in5 days 2 7 days (mg./kg.) Group volvement in ceca of chickens in an uninfectExperiment 1: a ed, unmedicated control. In Experiment 2, a 4.0 thiopeptin 2 4.0 Inf. E. tenella resistant to amprolium plus ethopa4.0 a 20 4.0 a bacitracin Inf. 4.0 a penicillin bate caused severe cecal lesions in birds given 12 4.0 a Inf. 4.0 a chlortetracycline 22 4.0 a Inf. amprolium plus ethopabate alone, those given Inf. a combination of the coccidiostat and antibia a 4.0 4.0 None unmed. otic, and those received basal ration. Lesion Uninf. scores taken on 5 and 7 days after infection 0 None 0 unmed. among infected groups were not statistically Experiment 2: different (P > 0.05). No gross lesion was 4.0 a 125 4.0 a Inf. amprolium 8 ethopabate seen in birds of an uninfected, unmedicated 4.0 a 2 4.0 a Inf. thiopeptin control group.
1006
A. ARAKAWA AND O. OHE
Dose of antibiotics supplemented in the ration were levels used for improvement of performance in chickens. Thiopeptin (Mine et al., 1972), bacitracin, penicillin and chlortetracycline are active against gram-positive bacteria. Results of the study suggest that the concentration of the antibiotics in the cecal contents were above their minimum effective dose levels against C. perfringens and thus suppressed the increase of these organisms stimulated by coccidia infection. However, chlortetracycline which is also active against gram-negative bacteria did not suppress an increased population of Enterobacteriaceae excepting E. coli 7 days after infection in Experiment two. This could be dose-related; a higher dose of chlortetracycline could possibly have suppressed the Enterobacteriaceae. In spite of the fact that thiopeptin and bacitracin are not active against gram-negatives, thiopeptin suppressed the increase of Enterobacteriaceae, E. coli, and Aerobacter spp. and bacitracin reduced the count of E. coli 5 days after infection in Experiment two. These findings have to be reinvestigated since increased number of gram-negative bacteria in Experi-
ment one and those at 7 days after infection in Experiment two were not reduced by these antibiotics. Visco and Burns (1972a) and Bradley and Radhakrishnan (1973) have shown that C. perfringens and other bacteria are involved in the cecal coccidiosis syndrome in gnotobiotic chickens. Similar synergistic relationship between E. coli and E. brunetti was reported (Nagi and Mathey, 1972). Gross cecal lesions observed in the present study in antibiotic-treated infected chickens could be accounted for involvement of bacteria other than C. perfringens, because dietary antibiotics evidently did not reduce these organisms, especially E. coli. It is conceivable that under field conditions chickens are continuously exposed to both coccidia and bacteria. Antibiotic supplemented in the feed could possibly have some benefit in improving performance of chickens by suppressing C. perfringens in the ceca, particularly when drug-resistant coccidia are involved. ACKNOWLEDGEMENT The writers gratefully express their appreciation to Dr. K. Tsunoda of the National Institute of Animal Health, Tokyo, for sharing a strain of E. tenella; and Messrs. Matsubara and Takano and Misses Kita and Nomura for their technical assistance.
REFERENCES 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
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present study were 10 -3 - 5 and 10~6 on 5 and 7 days after infection, respectively. This disparate result could possibly be explained by differences in methods employed. It is of particular interest, however, that counts at 7 days after infection were greater than those at 5 days. Infection of coccidia also resulted in an increase of a total population of Enterobacteriaceael days after infection. This increase is thought to be largely contributed by higher counts of E. coli. These findings agree with observation by Hein and Timms (1972) and Bradley and Radhakrishnan (1973) that coccidia infection stimulated the growth of E. coli. Interesting findings are that total counts of Enterobactericeae and number of E. coli and Aerobacter spp. at 7 days after infection were greater than those at 5 days.
ANTIBIOTICS AND CLOSTRIDIUM
1007
tativen Analyse der Darmflora von Menschen und Tieren. Zbl. Bakt. I. Abt. Orig. 195: 455-469. Nagi, M. S., and W. J. Mathey, 1972. Interaction of Escherichia coliand Eimeria brunettiin chickens. Avian Dis. 16: 864-873. Radhakrishnan, C. V., and R. E. Bradley, 1973. Comparative pathology and lesions of experimental infections with Eimeria tenella in germ-free, specific pathogen-free and conventional chickens. In: Germfree Research: Biological Effect of Gnotobiotic Environments, Ed. J. B. Heneghan, Academic Press, New York, N. Y., p. 451-455. Rohde, P. A., 1970. BBL Manual of Products and Laboratory Procedures, 5th Ed., BBL, Maryland, 211 P. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York, N. Y. 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.
NEWS AND NOTES (Continued from page 991) Staff Scientist, Poultry, Livestock and Veterinary Sciences, previously was Veterinary Medical Officer, Regional Poultry Research Laboratory, U.S. Department of Agriculture, East Lansing, Michigan. He was also an Associate Professor of Microbiology at Michigan State University, East Lansing.
Regional FFA competition during the Eastern States Exposition at West Srpingfield, Massachusetts. Richard DeGrange led the team, finishing as high individual in the contest. Other team members were his twin brother, Robert DeGrange, Dorothy Davis and David Male. They were coached by Dale White, a Vocational Agriculture teacher at South Hagerstown.
WOLVERINE NOTES Norm Clizer, Sales Consultant, Wolverine Manufacturing Company, Grand Haven, Michigan, has moved from Evansville, Indiana, to 61 Tamworth Circle, Bella Vista, Arkansas 72712. He will be responsible for contacting large commercial poultrymen in the southeastern and southwestern states. MARYLAND NOTES The team from South Hagerstown (Washington County) High School placed second in the Maryland FFA poultry judging contest, May 2, at the University of Maryland, College Park. Four and a half months later they repeated in the 12-state North Atlantic
A.I.B.S. NOTES Richard Trumbull was appointed Executive Director of the American Institute of Biological Sciences, Arlington, Virginia, on October 1, 1974, filling the vacancy caused by the death of John R. Olive on March 30. Trumbull served most recently as Deputy Executive Director of the American Association for the Advancement of Science, after being Director of the Office of Naval Research. He taught at Green Mountain Junior College, and at Syracuse University where he received a Ph.D. degree, and was a Lecturer at the University of Maryland and at Tulane University. He served in the U.S. Navy during World War
(Continued on page 1018)
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infected with Eimeria tenella. J. Bacteriol. 56: 635647. 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. Johnson, J., W. M. Reid and R. L. Kemp, 1973. The development of Eimeria tenella in germfree chickens. In: Germfree Research: Biological Effect of Gnotobiotic Environments, Ed. J. B. Heneghan, Academic Press, New York, N. Y., p. 457-460. Lafont, J. P., 1966. Flora intestinale et parasitoses: l'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. Mine, K., N. Miyairi, N. Takano, S. Mori and N. Watanabe, 1972. Thiopeptin, a new feed-additive antibiotic: Biological studies and field trials. Antimicob. Ag. Chemother. 1: 496-503. Mitsuoka, T., T. Sega and S. Yamamoto, 1965. Eine verbesserte Methodik der qualitativen und quanti-
PERFRINGENS
W. A. BOUGH, A. L. SHEWFELT AND W. L. SALTER
Mercer, W. A., 1969. A guide for waste management in the food processing industries. National Canners Assoc, Berkeley, Calif. Peniston, Q. P., and E. L. Johnson, 1970. Method for treating an aqueous medium with chitosan and derivatives of chitin to remove an impurity. U.S. Patent No. 3,533,940. Perceval, P. M., 1974. Personal communication, December 18. Singh, S. P., R. L. Wesley and E. A. Budd, 1973. Characteristics of poultry processing effluent. Poultry Sci. 52: 1478-1481. Whitehead, W. K., 1974. Analysis of some physical properties of poultry processing chiller effluent. Poultry Sci. 53: 571-573.
Reduction of Clostridium perfringens by Feed Additive Antibiotics in the Ceca of Chickens Infected with Eimeria tenella A. ARAKAWA AND O. OHE
Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 2-1-6, Kasima-cho, Yodogawa-ku, Osaka 532, Japan (Received for publication September 17, 1974)
ABSTRACT Two experiments were performed to investigate the effect of feed additive antibiotics on Clostridium perfringens and Enterobacteriaceae in the ceca of chickens infected with Eimeria tenella. In the first experiment, chickens were continuously fed rations containing thiopeptin, 2 mg./kg.; bacitracin, 20 mg./kg.; penicillin, 12 mg./kg.; or chlortetracycline, 22 mg./kg. One day after antibiotic feed was given, each bird received an oral inoculation of 30,000 E. tenella oocysts. The growth of C. perfringens was stimulated by the infection in unmedicated chickens. Dietary thiopeptin, bacitracin, penicillin, or chlortetracycline suppressed the number of C. perfringens recovered 5 and 7 days after infection. Enterobacteriaceae were increased by the infection, but dietary antibiotics did not reduce the increase. In the second experiment, chickens were given feed containing amprolium plus ethopabate, 125 plus 8 mg./kg., and a combination of the coccidiostat and one of 4 antibiotics: thiopeptin, bacitracin, penicillin, or chlortetracycline. Birds were each given an oral inoculation of 30,000 coccidiostat-resistant E. tenella oocysts. Infection resulted in an increase of C. perfringens in the unmedicated control and the coccidiostat-treated groups. Dietary thiopeptin, bacitracin, penicillin, or chlortetracycline reduced the number of C. perfringens found 5 and 7 days after infection. Counts of Enterobacteriaceae were increased by the infection, but dietary antibiotics did not suppress the increased counts. In both experiments, dietary administration of antibiotics did not reduce gross cecal lesions. POULTRY SCIENCE 54: 1000-1007, 1975
INTRODUCTION
C
HANGES in the bacterial flora of the
intestinal tract of chickens infected with
enterococci in the ceca of chickens infected with Eimeria tenella. Similar findings were observed by Lafont (1966) and Bradley and
coccidia have been reported. Johansson and
Radhakrishnan
Sarles (1948) found an increase of Clostridium
(1972) reported a significant increase of Es-
(1973).
Hein
and
Timms
perfringens and decrease of lactobacilli and
cherichia coli and C. perfringens in the small
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CRESA (Food, Chemical and Research Laboratories, Inc. and Engineering Science of Alaska), 1971. Pollution abatement and by-product recovery in shellfish and fisheries processing. Environmental Protection Agency, Project No. 12130FJQ, Supt. of Documents, Washington, D.C. Culp, R. L., and G. L. Culp, 1971. Advanced Wastewater Treatment. VanNostrand Reinhold Company, New York, N.Y., p. 256. Hamm, D., 1972. Characteristics of effluents in ten Southeastern poultry processing plants. Poultry Sci. 51:825-829. Mauldin, A. F., 1974. Case study—treatment of Gulf shrimp processing and canning wastes. Environmental Protection Agency, Technology Transfer Seminar, New Orleans, La., March 5-6.
ANTIBIOTICS AND CLOSTRIDIUM PERFRINGENS
TABLE 1.—Composition of basal ration Ingredients % Ground yellow corn 51.40 Soybean meal 20.00 Ground milo 14.00 Fish meal 8.00 Alfalfa meal 3.00 Calcium carbonate 1.50 Tricalcium phosphate 1.00 Salt 0.50 DL-methionine 0.20 Micronutrients' 0.40 1 Micronutrients provided in mg. and in units per kg. of ration: thiamine, 2 mg.; riboflavin, 10 mg.; pyridoxine, 1 mg.; niacin, 6 mg.; calcium pantothenate, 6.5 mg.; choline chloride, 120 mg.; folic acid, 0.2 mg.; vitamin A, 10,000 U.; vitamin D 3 , 2,000 U.; vitamin E, 10 mg.; manganese, 166 mg.; iron, 60 mg.; copper, 6 mg.; cobalt, 0.2 mg.; zinc, 30 mg.
MATERIALS AND METHODS Birds. White Leghorn, Hy-Line®, cockerels were obtained from a local commercial hatchery when less than one day old. They were reared in conventional electrically heated battery brooders and fed the basal ration until use. They were caged in batteries in air-conditioned rooms with continuous artificial illumination. Diets. The composition of the basal ration is given in Table 1. Medicated feeds were prepared by mixing the basal ration with a measured amount of commerical 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; and amprolium plus ethopabate, 5 125 plus 8. Experiment 1. A total of 60 11-day-old birds, averaging 97 g. of body weight, were divided into 6 groups of 10 chickens each. The groups consisted of those infected fed a ration containing either thiopeptin, bacitracin, penicillin, or chlortetracycline. The two groups were an infected, unmedicated group and an uninfected, unmedicated group. The respective rations and water were given ad libitum beginning one day prior to coccidia exposure until 7 days after infection. A strain of E. tenella used was maintained by Dr. K. Tsunoda of the National Institute of Animal Health in Tokyo and supplied to this laboratory. Fresh oocyst cultures were prepared routinely from donor chickens 7 to 8 days after infection. Oocysts were washed with sterile saline solution six times immediately before use and 0.1 ml. of the suspension was
1. 2. 3. 4. 5.
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.
Downloaded from http://ps.oxfordjournals.org/ at University of Arizona on June 7, 2016
intestine and ceca of chickens infected with E. brunetti. In gnotobiotic chickens, Visco and Burns (1972b) and Bradley and Radhakrishnan (1973) demonstrated that not only C. perfringens but also other bacteria including E. coli were responsible in part for the development of cecal coccidiosis. Recent studies in bacteria-free and conventional chicks have shown that germ-free chicks were less susceptible than conventionals to cecal coccidiosis (Visco and Burns, 1972a; Radhakrishnan and Bradley, 1973) and that gross lesions in germ-free chickens were less severe than those found in conventional infected chickens (Johnson et al., 1973). These findings and prevalent use of antibiotics in the current poultry feed stimulated the investigators to determine if antibiotics given via the feed would suppress the increase of C. perfringens stimulated by E. tenella infection and influence gross lesions. At the same time, a similar investigation was directed toward Enterobacteriaceae. Two studies were made: E. tenella infection in the presence of antibiotics in the feed and coccidiostat-resistant E. tenella infection in the presence of antibiotics and coccidiostat.
1001
1002
A. ARAKAWA AND O. OHE
Experiment 2. A total of 70 chickens, 11 days old, were allotted into 7 groups of 10 birds each. The groups consisted of those infected fed a ration containing amprolium plus ethopabate, and a combination of the coccidiostat and one of 4 antibiotics, i.e., thiopeptin, bacitracin, penicillin, or chlortetracycline. The two groups were an infected, unmedicated group and an uninfected, unmedicated group. The respective rations and water were available ad libitum starting one day before coccidia exposure until 7 days after infection. The E. tenella used was a coccidiostat-resistant strain which was obtained from litter samples from a local broiler house, separated by a single-oocyst method, and propagated through chickens under strictly isolated conditions. This strain was tested previously by the methods described by McManus et al. (1968) and found to be resistant to amprolium plus ethopabate, 125 plus 8 mg./kg., in the feed (the anticoccidial index = 121). Oocysts were washed with sterile saline six times before exposure and tested for sterility. Chickens in infected groups each received a single oral dose of 30,000 sporulated oocysts. Necropsy procedures were the same as those in the previous experiment. Bacteriological
Examination,
CW agar (5
g. of heart muscle infusion (Nissan), 10 g. of proteose peptone W (Nissan), 10 g. of casein peptone (Nissan), 5 g. of sodium chloride, 10 g. of lactose, 0.05 g. of phenol red, 20 g. of agar, and 0.2 g. of kanamycin sulfate per 1,000 ml. of distilled water; Nissui Kagaku Co., Ltd., Tokyo) was used to recover C. perfringens. DHL agar (Eiken Kagaku Co., Ltd., Tokyo) was used to count a total population of Enterobacteriaceae (Mitsuoka et al., 1965). EMB agar was utilized to enumerate E. coli and Aerobacter spp. Oocyst suspensions used for infecting chickens were tested for sterility by spreading 0.1 ml. over CW and DHL plates. At necropsy, ceca were isolated and midportion was opened under aseptic procedures. Cecal samples from a single animal were placed in a weighed and sterilized anaerobic glass tube containing approximately 120 glass beads (2.5 mm. in diameter). Tubes were weighed and the initial dilution was made by adding 9 volumes of sterile anaerobic diluent (4.5 g. of K H 2 P 0 4 , 6.0 g. of N a 2 H P 0 4 , 0.5 g. of L-cysteine-HCl • H 2 0 , 0.5 g. of Tween 80, 1.0 g. of agar and 1,000 ml. of distilled water). The tube was shaken until a homogenized suspension was obtained. One ml. of the suspension was withdrawn and further diluted in sterile anaerobic diluent by serial 10-fold steps. From each of the serial dilutions including the initial dilution, 0.1 ml. of suspension was taken, placed and spread on CW agar. Plates were incubated anaerobically in a Gaspak jar (with disposable hydrogen plus carbon dioxide generator envelopes, BBL) at 37° C. for 48 hours. Colonies characterized by lecithinase production were counted. A 0.1 ml. of suspension from each of the serial dilutions was spread over DHL and EMB agar plates. They were incubated aerobically at 37° C. for 24 hours. Colonies of E. coli characterized by a convex surface of 3 to 4 mm. in diameter appearing blackmetallic gold were counted. For identification, TSI agar, SIM medium, and Voges-
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provided for bacteriological examination. Birds in infected groups were each given an oral inoculation of 30, 000 sporulated oocysts. Five chickens in each group were killed 5 days after infection and the remaining animals were killed 2 days later for gross observation of the ceca and the bacteriological examination. At necropsy, cecal lesion scores were graded as described by Johnson and Reid (1970). They used a 0 to 4 scoring system with 1 for very few lesions, 2 for slight lesions, 3 for many lesions and considerable blood, and 4 for severe lesions and large amount of blood.
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Experiment 2. Table 3 contains the results. The counts of C. perfringens were significantly increased by a coccidiostat-resistant E. tenella infection in infected controls and
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Experiment I. Results are presented in Table 2. The increased number of C. perfringens detected 5 and 7 days after E. tenella infection in non-antibiotic treated controls was suppressed significantly by dietary administration of thiopeptin, bacitracin, penicillin, or chlortetracycline (P < 0.05). Enterobacteriaceae were increased by the coccidia infection; the increase was statistically significant only 7 days after infection (P < 0.05), but dietary antibiotics did not suppress the increase (P > 0.05). The growth of E. coli was similarly stimulated by the infection 5 and 7 days after infection, but the growth was statistically significant only 7 days after infection (P < 0.05). However, the growth was not suppressed by antibiotics in the diet (P > 0.05). There were no statistical differences in counts of Aerobacter spp. among 6 groups of chickens killed 5 and 7 days after infection (P > 0.05). No colonies were found on CW and DHL agar plates that were streaked with the oocyst suspension used for the infection.
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Statistical Analysis. Data on number of bacteria after square root transformation and cecal lesion scores were submitted to analysis of variance and Duncan's new multiple range test (P = 0.05) (Steel and Torrie, 1960).
D.
5 days-
Proskauer broth were used (Rohde, 1970). Colonies of Aerobacter spp. were counted. They were characterized by a concave surface of 4 to 6 mm. in diameter with brown centers. When no colony was found on agar plates with the initial dilution of suspension, the count was expressed as less than 100 per g. of cecal contents.
PERFRINGENS
ays
ANTIBIOTICS AND CLOSTRIDIUM
amprolium ethopabate thiopeptin amprolium ethopabate bacitracin amprolium ethopabate penicillin amprolium ethopabate chlortetracycline amprolium ethopabate
125 8 2 125 8 20 125 8 12 125 8 22 125 8 <10 2 a
<102
<10 2 a
<10 2 a
<10 2 a
<10 2 a
<10 2 a
OO23
2.7 x 108a
4.0 x 108a
1.9 x 10 8ab
6.3 x 107bc
2.9 x 109ab
2.5 x 109ab
4.4 x 109ab
2.7 x 109ab
1.0 x 10
1.4 x 10
2.5 x 10
2.4 x 10
Average number of organisms/g. of c Enterobacteriaceae 5 days 7 days 5 day 4.0 x 108a 1.8 x 109b 8.4 x 10
Inf. ;.6 x 106 3.6 x 108a 8.6 x 109a 4.0 x 10 unmed. None 1.2 x 103 Uninf. None unmed. <10 2 a <10 2 a 2.1 x 107c 5.2 x 107 7.0 x 10 '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.
Group Inf.
C. perfringens 5 days 7 days 3.4 x 104 :.2 x 107
of feed additive antibiotics on C. perfringens and Enterobacteriaceae in the ceca of strain of E. tenella (Experiment 2)
Supplement in feed (mg./kg.)
TABLE 3.—Effect
ded from http://ps.oxfordjournals.org/ at University of Arizona on June 7, 2016
ANTIBIOTICS AND CLOSTRIDIUM PERFRINGENS
in infected and amprolium plus ethopabate treated birds (P < 0.05). Dietary antibiotics combined with the coccidiostat prevented the increase of C. perfringens (P < 0.05). A total Enterobacteriaceae population was increased by the coccidia infection in non-antibiotic treated controls and in amprolium plus ethopabate medicated birds 5 and 7 days after exposure (P < 0.05). This increase was suppressed by dietary thiopeptin only 5 days after infection. E. coli was increased by coccidia infection, but significant increase was noted
1005
only 7 days after infection (P < 0.05). Dietary thiopeptin and bacitracin reduced the counts 5 days after infection and chlortetracycline suppressed E. coli 7 days after infection. Aerobacter spp. were significantly increased by coccidia infection (P < 0.05). Dietary thiopeptin reduced the increase only 5 days after infection. No colonies were formed on CW and DHL agar plates streaked with the oocyst inocula used.
Inf.
Inf.
Inf. Inf. unmed. Uninf. unmed.
amprolium ethopabate bacitracin amprolium ethopabate penicillin amprolium ethopabate chlortetracycline amprolium ethopabate
125 8 20 125 8 12 125 8 22 125 8
4.0 a
4.0 a
4.0 a
4.0 a
4.0 a
4.0 a
None
4.0 a
4.0 a
None
0
0
'Within each column, any averages having the same superscript are not significantly different at 0.05 level of probability. 2 Days after coccidia infection.
DISCUSSION The present study confirms the findings that the growth of C. perfringens in the ceca was stimulated by E. tenella infection (Johansson and Sarles, 1948; Bradley and Radhakrishnan, 1973) and demonstrates that the growth was equally stimulated by a coccidiostat-resistant E. tenella infection in the presence of coccidiostat in the feed. Johansson and Sarles (1948) found C. perfringens -like colonies up to 10" 8 dilution on the 5th day of infection although no counts were reported on the 7th day, whereas the counts in the
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Gross Pathology. Table 4 summarizes gross cecal lesion scores obtained 5 and 7 days after infection. In Experiment 1, a sublethal TABLE 4.—Effect of feed additive antibiotics on dose of coccidia resulted in severe lesions gross lesions of the ceca of chickens infected with with large volume of blood in ceca of all E. tenella (Experiments 1 and 2) birds in infected groups. Lesion scores among Supplement these groups were not significantly different i \ \ . lesion score' in feed (P > 0.05). There was no pathological in5 days 2 7 days (mg./kg.) Group volvement in ceca of chickens in an uninfectExperiment 1: a ed, unmedicated control. In Experiment 2, a 4.0 thiopeptin 2 4.0 Inf. E. tenella resistant to amprolium plus ethopa4.0 a 20 4.0 a bacitracin Inf. 4.0 a penicillin bate caused severe cecal lesions in birds given 12 4.0 a Inf. 4.0 a chlortetracycline 22 4.0 a Inf. amprolium plus ethopabate alone, those given Inf. a combination of the coccidiostat and antibia a 4.0 4.0 None unmed. otic, and those received basal ration. Lesion Uninf. scores taken on 5 and 7 days after infection 0 None 0 unmed. among infected groups were not statistically Experiment 2: different (P > 0.05). No gross lesion was 4.0 a 125 4.0 a Inf. amprolium 8 ethopabate seen in birds of an uninfected, unmedicated 4.0 a 2 4.0 a Inf. thiopeptin control group.
1006
A. ARAKAWA AND O. OHE
Dose of antibiotics supplemented in the ration were levels used for improvement of performance in chickens. Thiopeptin (Mine et al., 1972), bacitracin, penicillin and chlortetracycline are active against gram-positive bacteria. Results of the study suggest that the concentration of the antibiotics in the cecal contents were above their minimum effective dose levels against C. perfringens and thus suppressed the increase of these organisms stimulated by coccidia infection. However, chlortetracycline which is also active against gram-negative bacteria did not suppress an increased population of Enterobacteriaceae excepting E. coli 7 days after infection in Experiment two. This could be dose-related; a higher dose of chlortetracycline could possibly have suppressed the Enterobacteriaceae. In spite of the fact that thiopeptin and bacitracin are not active against gram-negatives, thiopeptin suppressed the increase of Enterobacteriaceae, E. coli, and Aerobacter spp. and bacitracin reduced the count of E. coli 5 days after infection in Experiment two. These findings have to be reinvestigated since increased number of gram-negative bacteria in Experi-
ment one and those at 7 days after infection in Experiment two were not reduced by these antibiotics. Visco and Burns (1972a) and Bradley and Radhakrishnan (1973) have shown that C. perfringens and other bacteria are involved in the cecal coccidiosis syndrome in gnotobiotic chickens. Similar synergistic relationship between E. coli and E. brunetti was reported (Nagi and Mathey, 1972). Gross cecal lesions observed in the present study in antibiotic-treated infected chickens could be accounted for involvement of bacteria other than C. perfringens, because dietary antibiotics evidently did not reduce these organisms, especially E. coli. It is conceivable that under field conditions chickens are continuously exposed to both coccidia and bacteria. Antibiotic supplemented in the feed could possibly have some benefit in improving performance of chickens by suppressing C. perfringens in the ceca, particularly when drug-resistant coccidia are involved. ACKNOWLEDGEMENT The writers gratefully express their appreciation to Dr. K. Tsunoda of the National Institute of Animal Health, Tokyo, for sharing a strain of E. tenella; and Messrs. Matsubara and Takano and Misses Kita and Nomura for their technical assistance.
REFERENCES 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
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present study were 10 -3 - 5 and 10~6 on 5 and 7 days after infection, respectively. This disparate result could possibly be explained by differences in methods employed. It is of particular interest, however, that counts at 7 days after infection were greater than those at 5 days. Infection of coccidia also resulted in an increase of a total population of Enterobacteriaceael days after infection. This increase is thought to be largely contributed by higher counts of E. coli. These findings agree with observation by Hein and Timms (1972) and Bradley and Radhakrishnan (1973) that coccidia infection stimulated the growth of E. coli. Interesting findings are that total counts of Enterobactericeae and number of E. coli and Aerobacter spp. at 7 days after infection were greater than those at 5 days.
ANTIBIOTICS AND CLOSTRIDIUM
1007
tativen Analyse der Darmflora von Menschen und Tieren. Zbl. Bakt. I. Abt. Orig. 195: 455-469. Nagi, M. S., and W. J. Mathey, 1972. Interaction of Escherichia coliand Eimeria brunettiin chickens. Avian Dis. 16: 864-873. Radhakrishnan, C. V., and R. E. Bradley, 1973. Comparative pathology and lesions of experimental infections with Eimeria tenella in germ-free, specific pathogen-free and conventional chickens. In: Germfree Research: Biological Effect of Gnotobiotic Environments, Ed. J. B. Heneghan, Academic Press, New York, N. Y., p. 451-455. Rohde, P. A., 1970. BBL Manual of Products and Laboratory Procedures, 5th Ed., BBL, Maryland, 211 P. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York, N. Y. 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.
NEWS AND NOTES (Continued from page 991) Staff Scientist, Poultry, Livestock and Veterinary Sciences, previously was Veterinary Medical Officer, Regional Poultry Research Laboratory, U.S. Department of Agriculture, East Lansing, Michigan. He was also an Associate Professor of Microbiology at Michigan State University, East Lansing.
Regional FFA competition during the Eastern States Exposition at West Srpingfield, Massachusetts. Richard DeGrange led the team, finishing as high individual in the contest. Other team members were his twin brother, Robert DeGrange, Dorothy Davis and David Male. They were coached by Dale White, a Vocational Agriculture teacher at South Hagerstown.
WOLVERINE NOTES Norm Clizer, Sales Consultant, Wolverine Manufacturing Company, Grand Haven, Michigan, has moved from Evansville, Indiana, to 61 Tamworth Circle, Bella Vista, Arkansas 72712. He will be responsible for contacting large commercial poultrymen in the southeastern and southwestern states. MARYLAND NOTES The team from South Hagerstown (Washington County) High School placed second in the Maryland FFA poultry judging contest, May 2, at the University of Maryland, College Park. Four and a half months later they repeated in the 12-state North Atlantic
A.I.B.S. NOTES Richard Trumbull was appointed Executive Director of the American Institute of Biological Sciences, Arlington, Virginia, on October 1, 1974, filling the vacancy caused by the death of John R. Olive on March 30. Trumbull served most recently as Deputy Executive Director of the American Association for the Advancement of Science, after being Director of the Office of Naval Research. He taught at Green Mountain Junior College, and at Syracuse University where he received a Ph.D. degree, and was a Lecturer at the University of Maryland and at Tulane University. He served in the U.S. Navy during World War
(Continued on page 1018)
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infected with Eimeria tenella. J. Bacteriol. 56: 635647. 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. Johnson, J., W. M. Reid and R. L. Kemp, 1973. The development of Eimeria tenella in germfree chickens. In: Germfree Research: Biological Effect of Gnotobiotic Environments, Ed. J. B. Heneghan, Academic Press, New York, N. Y., p. 457-460. Lafont, J. P., 1966. Flora intestinale et parasitoses: l'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. Mine, K., N. Miyairi, N. Takano, S. Mori and N. Watanabe, 1972. Thiopeptin, a new feed-additive antibiotic: Biological studies and field trials. Antimicob. Ag. Chemother. 1: 496-503. Mitsuoka, T., T. Sega and S. Yamamoto, 1965. Eine verbesserte Methodik der qualitativen und quanti-
PERFRINGENS