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Effect of Eimeria tenella infection on the caecal population of lincomycin-resistant Clostridium perfringens introduced into chickens
Research in Veterinary Science /988, 45, 2/9-22/
Effect of Eimeria tenella infection on the caecal population of lincomycin-resistant Clostridium perfringens introduced into chickens E. BABA, N. YASUDA, T. FUKATA, A. ARAKAWA, Department of Veterinary Medicine, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 591, Japan The effect of Eimeria tenella infection on the caecal population of lincomycin-resistant Clostridium perfringens (KGW 851) newly introduced into chickens was studied. Four groups of chickens consisted of: (I) Uninoculated controls; (2) inoculated with C perfringens (KGW 851); (3) inoculated with E tenella; and (4) inoculated with C perfringens (KGW 851) followed by E tenella. Five chickens in each group were necropsied on each of days 0,3,5,7, 10 and 14 after the E tenella inoculation. The mean total C perfringens counts in the caecal contents increased at five days after E tenella inoculation and reached maximum counts at seven days after the inoculation. Also Iincomycinresistant C perfringens readily established itself at approximately one 10th of the total C perfringens population in the presence or absence of E tenella infection. THE object.of this study was to examine the establishment of a newly introduced Clostridium perfringens resistant to lincomycin (KGW 851) in the caeca, and the effect of concurrent Eimeria tenella infection on caecal clostridial counts in chickens. Materials and methods
Birds Day-old White Leghorn Hy-Line cockerels from a local commercial hatchery were kept in steamdisinfected cages with a metal plate floor, in an air conditioned room with filtered ventilation and continuous artificial illumination. Birds were given a basal ration (Arakawa and Ohe 1975) and water ad libitum during the study.
organism was grown in thioglycollate broth (Nissui, Tokyo) at 37°C for 18 hours. Eimeria ten ella The strain of E tenella was isolated by the National Institute of Animal Health, Tsukuba, Japan. Sporulated oocysts were prepared from faeces of donor chickens seven to eight days after oral inoculation.
Bacteriological examination Clostridium welchii medium (Nissui) was prepared for determination of total counts of C perfringens, while C welchii medium containing 100 j.lg ml'! of lincomycin was used as selective medium for quantitation of lincomycin-resistant C perfringens. At necropsy, caeca were isolated from each bird and their contents were weighed and placed into a glass tube. The initial dilution of caecal contents was made by adding nine volumes of sterile phosphate buffered' saline (PBS, pH 7· 2) with reducing agents (Mitsuoka et al 1965). Additional serial IO-fold dilutions were made in PBS. From each of the dilutions, including the initial dilution, 0'01 ml was withdrawn and spread on to one third part of each agar plate. The agar plates were incubated in anaerobic jars (Anaerobic System, Oxoid) at 37°C for 24 hours. Rough, brownish colonies showing lecithinase production were presumptively identified as C perfringens. Numbers of the organisms were expressed as 10gIO of colony forming units (CFU) g-I of caecal contents. The limit of C perfringens detection was 3 10gIO g -I of caecal contents. Experimental design
Clostridium perfringens The C perfringens strain, KGW 851 (C perfringens type A [McDonel 1985]), was isolated from a 15-week-old broiler chicken in a field outbreak of necrotic enteritis. The bird was hatched from an egg imported from the USA. The organism was resistant to 200 J.Ag rnl ' ' lincomycin. For inoculation, the
Three trials were conducted. In each trial, a total of 120, day-old chickens were divided into four groups of 30 chickens each. The experimental groups were: an uninoculated control, a group inoculated with lincomycin-resistant C perfringens, a group inoculated with E tenella, and a group inoculated with both lincomycin-resistant C perjringens and E
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E. Baba, N. Yasuda, T. Fukata, A. Arakawa
tenella. Chickens in the two C perjringens-inoculated groups received a daily oral inoculation of lOS to 1()6 CFU of the C perjringens at one to three days of age. Birds in the two E tenella-inoculated groups were each given an oral dose of 2x 1()4 (trials 1 and 2) or 4 x 1()4 (trial 3) sporulated oocysts in 1 ml of tap water at the age of five days. Five chickens in each group were necropsied on the day of E tenella inoculation and three, five, seven, 10 and 14 days after E tenella inoculation for gross pathological and bacteriological examinations. Statistical analysis The number of C perjringens was expressed as 10glO and data were subjected to analysis of variance and Duncan's new multiple range test (Steel and Torrie 1960). Results No gross lesions of avian necrotic enteritis were observed and C perjringens inoculation had no effect on caecal lesions caused by E tenella. Total C perjringens counts in the caecal contents of chickens in each group are presented in Fig I as the mean bacterial counts in three trials. In the uninoculated control, the mean total C perjringens counts at the first necropsy was 5' 0 and was followed by a gradual decrease. In the group inoculated with lincomycin-resistant C perfringens, mean organism counts exhibited a similar decline, but the values at the first necropsy and those 14 days later were signifi8
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10
Days after Eimeria tenella inoculation
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FIG 1: Total Clostridium perfringens counts in the caecal contents of chickens inoculated with lincomycin-resistant C perfringens and Eimeria tenella. Groups consisted of non-inoculated control (0). birds inoculated with lincomycin-resistant C perfringens (01. birds inoculated with E tanella (e) and birds inoculated with both organisms I.). Means at the same necropsy day with the same superscript do not differ significantly I P>0·051
cantly (P<0'05) higher than those of uninoculated controls. In the two E tenella-inoculated groups the mean total C perjringens counts began to increase at five days after the E tenella inoculation, reaching a peak value seven days after the inoculation, decreasing thereafter. From five to 14 days, the mean total C perfringens counts of the E tenella infected groups were significantly (P<0·05) higher than those of E tenella uninoculated groups. Only on the day of E tenella inoculation was the mean total C perjringens count in the C perfringens-E tenella inoculated group similar to the group in which C perjringens was inoculated alone and significantly (P
Effect ofE tenella on C perfringens
Transferable antibiotic resistance in C perfringens strains has been reported. Fifteen of 89 tetracyclineresistant strains transferred their resistance in vitro, but transfer of microIide-lincosamide resistance was not detected (Rood 1983). However, the present data indicate that, even if lincomycin resistance of the C perfringens strain were transferred to susceptible C perfringens in the intestine, the mean total counts of lincomycin-resistant C perfringens would not be affected by the transfer. References ARAKAWA, A. & OHE, O. (1975) Poultry Science 54, 1000-1007 GEORGE, B. A., QUARLES, C. L. & FAGERBERG, D. J. (1982) Poultry Science 61, 447-450 HAMDY, A. H., THOMAS, R. W. & KRATZER, D. D. (l983a) Poultry Science 62, 585-588
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HAMDY, A. H., THOMAS, R. W. & YANCEY, R. J. (1983b) Poultry Science 62, 589-591 KIMURA, N., MIMURA, F., NISHIDA, S., KOBAYASHI, A. & MITSUOKA, T. (1976) Poultry Science 55, 1375-1383 MAXEY, B. W. & PAGE, R. K. (1977) Poultry Science 56, 1909-1913 McDONEl, J. L. (1985) Pharmacological Therapy 10,617-655 MITSUOKA, T., SEGA, T. & YAMAMOTO, S. (1965) Zentralblatt fur Bakteriotogle, Mikrobiologie und Hygiene IAI 195,455-469 ROOD. J. J. (1983) Canadian Journal of Microbiology 29, 12411246 STEEL, R. G. D. & TORRIE, J. H. (1960) Principles and Procedures of Statistics. New York. McGraw-Hili. pp 88-160 TRUSCOTT, R. B. & Al-SHEIKHlY, F. (1977)American Journal of Veterinary Research 38. 857-861 WICKER, D. L., ISGRIGG, W. N. & TRAMMEll, J. H. (1977) Poultry Science 56,1229-1231
Received June 30, 1987 Accepted October 21, 1987
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Effect of Eimeria tenella infection on the caecal population of lincomycin-resistant Clostridium perfringens introduced into chickens E. BABA, N. YASUDA, T. FUKATA, A. ARAKAWA, Department of Veterinary Medicine, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 591, Japan The effect of Eimeria tenella infection on the caecal population of lincomycin-resistant Clostridium perfringens (KGW 851) newly introduced into chickens was studied. Four groups of chickens consisted of: (I) Uninoculated controls; (2) inoculated with C perfringens (KGW 851); (3) inoculated with E tenella; and (4) inoculated with C perfringens (KGW 851) followed by E tenella. Five chickens in each group were necropsied on each of days 0,3,5,7, 10 and 14 after the E tenella inoculation. The mean total C perfringens counts in the caecal contents increased at five days after E tenella inoculation and reached maximum counts at seven days after the inoculation. Also Iincomycinresistant C perfringens readily established itself at approximately one 10th of the total C perfringens population in the presence or absence of E tenella infection. THE object.of this study was to examine the establishment of a newly introduced Clostridium perfringens resistant to lincomycin (KGW 851) in the caeca, and the effect of concurrent Eimeria tenella infection on caecal clostridial counts in chickens. Materials and methods
Birds Day-old White Leghorn Hy-Line cockerels from a local commercial hatchery were kept in steamdisinfected cages with a metal plate floor, in an air conditioned room with filtered ventilation and continuous artificial illumination. Birds were given a basal ration (Arakawa and Ohe 1975) and water ad libitum during the study.
organism was grown in thioglycollate broth (Nissui, Tokyo) at 37°C for 18 hours. Eimeria ten ella The strain of E tenella was isolated by the National Institute of Animal Health, Tsukuba, Japan. Sporulated oocysts were prepared from faeces of donor chickens seven to eight days after oral inoculation.
Bacteriological examination Clostridium welchii medium (Nissui) was prepared for determination of total counts of C perfringens, while C welchii medium containing 100 j.lg ml'! of lincomycin was used as selective medium for quantitation of lincomycin-resistant C perfringens. At necropsy, caeca were isolated from each bird and their contents were weighed and placed into a glass tube. The initial dilution of caecal contents was made by adding nine volumes of sterile phosphate buffered' saline (PBS, pH 7· 2) with reducing agents (Mitsuoka et al 1965). Additional serial IO-fold dilutions were made in PBS. From each of the dilutions, including the initial dilution, 0'01 ml was withdrawn and spread on to one third part of each agar plate. The agar plates were incubated in anaerobic jars (Anaerobic System, Oxoid) at 37°C for 24 hours. Rough, brownish colonies showing lecithinase production were presumptively identified as C perfringens. Numbers of the organisms were expressed as 10gIO of colony forming units (CFU) g-I of caecal contents. The limit of C perfringens detection was 3 10gIO g -I of caecal contents. Experimental design
Clostridium perfringens The C perfringens strain, KGW 851 (C perfringens type A [McDonel 1985]), was isolated from a 15-week-old broiler chicken in a field outbreak of necrotic enteritis. The bird was hatched from an egg imported from the USA. The organism was resistant to 200 J.Ag rnl ' ' lincomycin. For inoculation, the
Three trials were conducted. In each trial, a total of 120, day-old chickens were divided into four groups of 30 chickens each. The experimental groups were: an uninoculated control, a group inoculated with lincomycin-resistant C perfringens, a group inoculated with E tenella, and a group inoculated with both lincomycin-resistant C perjringens and E
219
220
E. Baba, N. Yasuda, T. Fukata, A. Arakawa
tenella. Chickens in the two C perjringens-inoculated groups received a daily oral inoculation of lOS to 1()6 CFU of the C perjringens at one to three days of age. Birds in the two E tenella-inoculated groups were each given an oral dose of 2x 1()4 (trials 1 and 2) or 4 x 1()4 (trial 3) sporulated oocysts in 1 ml of tap water at the age of five days. Five chickens in each group were necropsied on the day of E tenella inoculation and three, five, seven, 10 and 14 days after E tenella inoculation for gross pathological and bacteriological examinations. Statistical analysis The number of C perjringens was expressed as 10glO and data were subjected to analysis of variance and Duncan's new multiple range test (Steel and Torrie 1960). Results No gross lesions of avian necrotic enteritis were observed and C perjringens inoculation had no effect on caecal lesions caused by E tenella. Total C perjringens counts in the caecal contents of chickens in each group are presented in Fig I as the mean bacterial counts in three trials. In the uninoculated control, the mean total C perjringens counts at the first necropsy was 5' 0 and was followed by a gradual decrease. In the group inoculated with lincomycin-resistant C perfringens, mean organism counts exhibited a similar decline, but the values at the first necropsy and those 14 days later were signifi8
o
3
5
7
10
Days after Eimeria tenella inoculation
14
FIG 1: Total Clostridium perfringens counts in the caecal contents of chickens inoculated with lincomycin-resistant C perfringens and Eimeria tenella. Groups consisted of non-inoculated control (0). birds inoculated with lincomycin-resistant C perfringens (01. birds inoculated with E tanella (e) and birds inoculated with both organisms I.). Means at the same necropsy day with the same superscript do not differ significantly I P>0·051
cantly (P<0'05) higher than those of uninoculated controls. In the two E tenella-inoculated groups the mean total C perjringens counts began to increase at five days after the E tenella inoculation, reaching a peak value seven days after the inoculation, decreasing thereafter. From five to 14 days, the mean total C perfringens counts of the E tenella infected groups were significantly (P<0·05) higher than those of E tenella uninoculated groups. Only on the day of E tenella inoculation was the mean total C perjringens count in the C perfringens-E tenella inoculated group similar to the group in which C perjringens was inoculated alone and significantly (P
Effect ofE tenella on C perfringens
Transferable antibiotic resistance in C perfringens strains has been reported. Fifteen of 89 tetracyclineresistant strains transferred their resistance in vitro, but transfer of microIide-lincosamide resistance was not detected (Rood 1983). However, the present data indicate that, even if lincomycin resistance of the C perfringens strain were transferred to susceptible C perfringens in the intestine, the mean total counts of lincomycin-resistant C perfringens would not be affected by the transfer. References ARAKAWA, A. & OHE, O. (1975) Poultry Science 54, 1000-1007 GEORGE, B. A., QUARLES, C. L. & FAGERBERG, D. J. (1982) Poultry Science 61, 447-450 HAMDY, A. H., THOMAS, R. W. & KRATZER, D. D. (l983a) Poultry Science 62, 585-588
221
HAMDY, A. H., THOMAS, R. W. & YANCEY, R. J. (1983b) Poultry Science 62, 589-591 KIMURA, N., MIMURA, F., NISHIDA, S., KOBAYASHI, A. & MITSUOKA, T. (1976) Poultry Science 55, 1375-1383 MAXEY, B. W. & PAGE, R. K. (1977) Poultry Science 56, 1909-1913 McDONEl, J. L. (1985) Pharmacological Therapy 10,617-655 MITSUOKA, T., SEGA, T. & YAMAMOTO, S. (1965) Zentralblatt fur Bakteriotogle, Mikrobiologie und Hygiene IAI 195,455-469 ROOD. J. J. (1983) Canadian Journal of Microbiology 29, 12411246 STEEL, R. G. D. & TORRIE, J. H. (1960) Principles and Procedures of Statistics. New York. McGraw-Hili. pp 88-160 TRUSCOTT, R. B. & Al-SHEIKHlY, F. (1977)American Journal of Veterinary Research 38. 857-861 WICKER, D. L., ISGRIGG, W. N. & TRAMMEll, J. H. (1977) Poultry Science 56,1229-1231
Received June 30, 1987 Accepted October 21, 1987
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