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
Downloaded from http://ps.oxfordjournals.org/ at University of Arizona on June 7, 2016
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-
Downloaded from http://ps.oxfordjournals.org/ at University of Arizona on June 7, 2016
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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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
Downloaded from http://ps.oxfordjournals.org/ at University of Arizona on June 7, 2016
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-
Downloaded from http://ps.oxfordjournals.org/ at University of Arizona on June 7, 2016
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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