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Research Note: Population of Salmonella typhimurium in the Cecum of Gnotobiotic Chickens
Research Note: Population of Salmonella typhimurium Cecum of Gnotobiotic Chickens
in the
T. FUKATA, E. BABA, and A. ARAKAWA Department of Veterinary Medicine, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 591, Japan (Received for publication December 18, 1987)
1989 Poultry Science 68: 311-314 INTRODUCTION
Oral administration of suspensions of cecal contents or feces obtained from adult chickens to newly hatched chicks prevented subsequent colonization with salmonella (Nurmi and Rantala, 1973; Rigby et al., 1977; Snoeyenbos et al., 1978; Soerjadi et al., 1978; Barnes et al, 1980a,b; Impey et al., 1982). Administration of certain bacteria or defined mixtures of bacteria isolated from those suspensions also exerted a similar effect (Rigby et al., 1977; Soerjadi et al., 1978; Barnes et al., 1979, 1980a,b; Barrow and Tucker, 1986). Competitive exclusion of Salmonella typhimurium was reported in gnotobiotic chickens administered fecal suspensions from healthy adult hens or bacteria isolated from cecum of young chickens (Lafont et al., 1983; Hudault et al., 1986). Pure cultures of Lactobacillus acidophilus were effective for prophylactic and therapeutic treatment of pathogenic S. typhimurium (Watkins and Miller, 1983). The present study examined the effect of a single species of bacterium on levels of bacteria in gnotobiotic chickens subsequently inoculated with S. typhimurium. MATERIALS AND METHODS
Details of the germ-free chickens and diets used were given by Fukata et al. (1987a,b) and
Kageyama et al. (1987). The method of preparation of S. typhimurium. Strain L-55, was reported by Fukata et al. (1987a). Bacteroides vulgatus. Bifidobacterium thermophilum, Clostridium perfringens, and L. acidophilus were supplied by the Institute of Physical and Chemical Research in Saitama, Japan. A standard strain of Escherichia coli, O-150 antigen, was used (Fukata et al., 1987a, b; Kageyama et al., 1987). Media for bacteriological examination involving growth, selective media, saline, or buffer diluent were given by Fukata et al. (1987a,b). Methods of bacteriological examination were reported by Fukata et al. (1987b). Experimental Design. For the salmonella control group, chickens were each inoculated with S. typhimurium alone. For each test bacterium, chickens were placed in two groups; chickens in the test control group were inoculated with the test bacterium alone and in the combined group with both the test bacterium and S. typhimurium. Test bacteria were B. vulgatus, B. thermophilum, C. perfringens, L. acidophilus, and E. coli. At 2 days of age, chickens in the test (control) and combined groups were inoculated orally with 10 cfu of the test bacterium through a rat stomach tube passed through the isolator wall. Three days later, chickens in the salmonella control and combined groups were inoculated orally with 10 cfu of S. typhimurium.
311
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 9, 2015
ABSTRACT To test the interaction of various species of bacteria with Salmonella typhimurium, levels of S. typhimurium were measured in the cecum of gnotobiotic chickens inoculated with various test bacteria and subsequently with 5. typhimurium. The population of 5. typhimurium was suppressed in the cecum of chickens previously inoculated with Escherichia coli. Populations of Bacteroides vulgatus, Clostridium perfringens, Bifidobacterium thermophilum, or Lactobacillus acidophilus were transiently, though not significantly, suppressed by S. typhimurium. Inoculation of chickens with C. perfringens caused a terminal increase in the population of S. typhimurium. Thus the effect of cecal contents on reducing the levels of S. typhimurium in the cecum may be due to the presence of E. coli in die cecal contents. (Key words: Salmonella typhimurium, gnotobiotic chickens, ceca, bacteria population)
312
FUKATA ET AL. 10
10
5-
(a) S.
0
typhimurium
1
10
(c) B.
vulgatus I
o ,3
0
1
* 0
(d) L. *
acidophilus '
1 2
10
10
(e) C.
perfringens
(f) E. coli
0»>
0
6 8 0 1 2 4 6 8 Days after S. typhimurium exposure FIGURE 1(a). Salmonella typhimurium (control) chickens (n = 6 to 9). O = average count of S. typhimurium in the cecal contents of chickens inoculated with S. typhimurium alone (salmonella control); bar = standard deviation. Data presented are pooled. FIGURE l(b-f). Interaction between Salmonella typhimurium and test bacteria in the cecal contents. O = average count of S. typhimurium and • = average count of test bacterium in the chickens inoculated with the test bacterium and S. typhimurium (combined group); CI = average count of test bacterium in the chickens inoculated with the test bacterium alone (test control). Test bacterium: (b) Bifidobacterium thermophilum (n = 4); (c) Bacteroides vulgatus (n = 4-7); (d) Lactobacillus acidophilus (n = 4-5); (e) Clostridium perfringens (n = 4-6); and (f) Escherichia coli (n = 4-6). *Significantly different from S. typhimurium control (P<.05). ***Significantly different from S. typhimurium control (P<.001). 2
4
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 9, 2015
io r
313
RESEARCH NOTE
To assess the level of colonization, four to nine chickens in each group were killed at 1,2, 4, 6, and 8 days after S. typhimurium inoculation, and cecal contents were removed for bacteriological examination. The number of 5. typhimurium in cecal contents of salmonella control and combined groups was counted and the number of test bacteria in cecal contents of the test control and combined groups was also counted. Bacteriological counts were replicated twice. Statistical analysis was conducted using Student's / test. RESULTS AND DISCUSSION 8
9
typhimurium were between 10 and 10 cfu/g from 1 day after inoculation to the end of the experiment (Figure la). These counts were compared with those of S. typhimurium in the combined groups. In the combined groups inoculated with B. vulgatus and S. typhimurium, B. thermophilum and S. typhimurium, or L. acidophilus and S. typhimurium, counts of S. typhimurium were 10 to 10 cfu/g for the entire experimental period with the exception of 1 day after S. typhimurium inoculation in the L. acidophilus combined group (Figure la,b,c,d). These counts were similar to those of the salmonella control group (Figure la). In the combined group inoculated with C. perfringens and S. typhimurium, the counts of S. typhimurium at 8 days after inoculation were significantly greater (P<.05; Figure 2e) than in the salmonella control group. The counts of each test bacteria were lower at 1 day after S. typhimurium inoculation than before S. typhimurium inoculation (Figure lb, c,e) with the exception of the L. acidophilus combined group (Figure Id). The counts of L. acidophilus at 1 day after S. typhimurium inoculation were the same as the counts of L. acidophilus in the test control group. At 2 days after S. typhimurium inoculation, the counts of L. acidophilus decreased (Figure Id). In the combined group inoculated with E. coli and 5. typhimurium, the counts of S. typhimurium (Figure If) were lower than in the salmonella control group (Figure la) through the entire experimental period. The counts of E. coli were not influenced by 5. typhimurium inoculation. The counts of test bacteria remained low for at least two sampling periods before returning to approximately the same
REFERENCES Barnes, E. M , C. S. Impey, and D. M. Cooper, 1980a. Competitive exclusion of salmonellas from the newly hatched chicken. Vet. Rec. 106:61. Barnes, E. M , C. S. Impey, and D. M. Cooper, 1980b. Manipulation of the crop and intestinal flora of the newly hatched chick. Am. J. Clin. Nutr. 33:2426-2433. Barnes, E. ML, C. S. Impey, and B.J.H. Stevens, 1979. Factors affecting the incidences and anti-salmonella activity of the anaerobic caecal flora of the young chick. J. Hyg. 82:263-283. Barrow, P. A., and J. F. Tucker, 1986. Inhibition of colonization of the chicken caecum with Salmonella typhimurium by pre-treatment with strains of Escherichia coli. J. Hyg. 96:161-169. Fukata, T., E. Baba, and A. Arakawa, 1987a. Invasion of Salmonella typhimurium into the cecal wall of gnotobiotic chickens with Eimeria tenella. Poultry Sci. 66:760-761. Fukata, T„ A. Kageyama, E. Baba, and A. Arakawa, 1987b. Effect of infection with Eimeria tenella upon the cecal bacterial population in monoflora chickens. Poultry Sci. 66:841-844. Hudault, S., H. Bewa, C. Bridonneau, and C. Raibaud, 1985. Efficiency of various bacterial suspension derived from ceca flora of conventional chickens in reducing the population level of Salmonella typhimurium in gnotobiotic mice and chicken intestines. Can. J. Microbiol. 31:832-838. Impey, C. S., G. C. Mead, and S. M. George, 1982. Competitive exclusion of salmonellas from the chick caecum using a defined mixture of bacterial isolated from the caecal microflora of an adult bird. J. Hyg. 89:479-490. Kageyama, A., T. Fukata, E. Baba, and A. Arakawa, 1987. The influence of various bacteria on the cecal mucosa on monoflora chickens infected with Eimeria tenella. A scanning electron microscopic study. Zentralbl. Baktcriol. Mikrobiol. Hyg. Ser. A 265:353-359. Lafont, J. P., A. Bree, M. Naciri, P. Yvore, J. F. Guillot, and
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 9, 2015
In the salmonella control, the counts of S.
level as 1 day after S. typhimurium inoculation. In general, after combined exposure to 5. typhimurium and test bacteria, either the population of S. typhimurium, but not the test bacteria was suppressed, or the population of test bacteria, but not 5. typhimurium, was suppressed. Thus B. vulgatus, C. perfringens, B. thermophilus, and L. acidophilus temporarily was suppressed by S. typhimurium populations. This suppression, though not significant, was consistent within the four test bacteria. However, E. coli was not suppressed by S. typhimurium at 1 and 6 days after inoculation, a pattern that continued throughout the experiment. In one instance, C. perfringens appeared to enhance populations of S. typhimurium after 8 days. Escherichia coli may play a major role in suppressing populations of S. typhimurium in the cecum of chickens.
314
FUKATA EI At.
E. Chaslus-Dancla, 1983. Experimental study of some factors limiting 'competitive exclusion' of salmonella in chickens. Res. Vet. Sci. 34:16-20. Nurmi, E., and M. Rantala, 1973. New aspects of Salmonella infection in broiler production. Nature, London 241:210-211. Rigby, C. E., J. R. Pettit, and A. Robertson, 1977. The effect of normal intestinal flora on the Salmonella carrier state in poultry with special reference to S. thompson and 5. typhimurium. Page 263 in: Proc. Int. Symp. on
Salmonella and Prospects for Control. D. A. Barnum, ed. University of Guelph. Guelph, Ontario, Canada. Soerjadi, A. S., A. B. Lloyd, and R. B. Cumming, 1978. Streptococcus faecalis, a bacterial isolate which protects young chickens from enteric invasion by salmonellae. Aust. Vet. J. 54:549-550. Watkins, B. A., and B. F. Miller, 1983. Competitive gut exclusion of avian pathogens by Lactobacillus acidophilus in gnotobiotic chicks. Poultry Sci. 62:1772-1779.
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 9, 2015
in the
T. FUKATA, E. BABA, and A. ARAKAWA Department of Veterinary Medicine, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 591, Japan (Received for publication December 18, 1987)
1989 Poultry Science 68: 311-314 INTRODUCTION
Oral administration of suspensions of cecal contents or feces obtained from adult chickens to newly hatched chicks prevented subsequent colonization with salmonella (Nurmi and Rantala, 1973; Rigby et al., 1977; Snoeyenbos et al., 1978; Soerjadi et al., 1978; Barnes et al, 1980a,b; Impey et al., 1982). Administration of certain bacteria or defined mixtures of bacteria isolated from those suspensions also exerted a similar effect (Rigby et al., 1977; Soerjadi et al., 1978; Barnes et al., 1979, 1980a,b; Barrow and Tucker, 1986). Competitive exclusion of Salmonella typhimurium was reported in gnotobiotic chickens administered fecal suspensions from healthy adult hens or bacteria isolated from cecum of young chickens (Lafont et al., 1983; Hudault et al., 1986). Pure cultures of Lactobacillus acidophilus were effective for prophylactic and therapeutic treatment of pathogenic S. typhimurium (Watkins and Miller, 1983). The present study examined the effect of a single species of bacterium on levels of bacteria in gnotobiotic chickens subsequently inoculated with S. typhimurium. MATERIALS AND METHODS
Details of the germ-free chickens and diets used were given by Fukata et al. (1987a,b) and
Kageyama et al. (1987). The method of preparation of S. typhimurium. Strain L-55, was reported by Fukata et al. (1987a). Bacteroides vulgatus. Bifidobacterium thermophilum, Clostridium perfringens, and L. acidophilus were supplied by the Institute of Physical and Chemical Research in Saitama, Japan. A standard strain of Escherichia coli, O-150 antigen, was used (Fukata et al., 1987a, b; Kageyama et al., 1987). Media for bacteriological examination involving growth, selective media, saline, or buffer diluent were given by Fukata et al. (1987a,b). Methods of bacteriological examination were reported by Fukata et al. (1987b). Experimental Design. For the salmonella control group, chickens were each inoculated with S. typhimurium alone. For each test bacterium, chickens were placed in two groups; chickens in the test control group were inoculated with the test bacterium alone and in the combined group with both the test bacterium and S. typhimurium. Test bacteria were B. vulgatus, B. thermophilum, C. perfringens, L. acidophilus, and E. coli. At 2 days of age, chickens in the test (control) and combined groups were inoculated orally with 10 cfu of the test bacterium through a rat stomach tube passed through the isolator wall. Three days later, chickens in the salmonella control and combined groups were inoculated orally with 10 cfu of S. typhimurium.
311
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 9, 2015
ABSTRACT To test the interaction of various species of bacteria with Salmonella typhimurium, levels of S. typhimurium were measured in the cecum of gnotobiotic chickens inoculated with various test bacteria and subsequently with 5. typhimurium. The population of 5. typhimurium was suppressed in the cecum of chickens previously inoculated with Escherichia coli. Populations of Bacteroides vulgatus, Clostridium perfringens, Bifidobacterium thermophilum, or Lactobacillus acidophilus were transiently, though not significantly, suppressed by S. typhimurium. Inoculation of chickens with C. perfringens caused a terminal increase in the population of S. typhimurium. Thus the effect of cecal contents on reducing the levels of S. typhimurium in the cecum may be due to the presence of E. coli in die cecal contents. (Key words: Salmonella typhimurium, gnotobiotic chickens, ceca, bacteria population)
312
FUKATA ET AL. 10
10
5-
(a) S.
0
typhimurium
1
10
(c) B.
vulgatus I
o ,3
0
1
* 0
(d) L. *
acidophilus '
1 2
10
10
(e) C.
perfringens
(f) E. coli
0»>
0
6 8 0 1 2 4 6 8 Days after S. typhimurium exposure FIGURE 1(a). Salmonella typhimurium (control) chickens (n = 6 to 9). O = average count of S. typhimurium in the cecal contents of chickens inoculated with S. typhimurium alone (salmonella control); bar = standard deviation. Data presented are pooled. FIGURE l(b-f). Interaction between Salmonella typhimurium and test bacteria in the cecal contents. O = average count of S. typhimurium and • = average count of test bacterium in the chickens inoculated with the test bacterium and S. typhimurium (combined group); CI = average count of test bacterium in the chickens inoculated with the test bacterium alone (test control). Test bacterium: (b) Bifidobacterium thermophilum (n = 4); (c) Bacteroides vulgatus (n = 4-7); (d) Lactobacillus acidophilus (n = 4-5); (e) Clostridium perfringens (n = 4-6); and (f) Escherichia coli (n = 4-6). *Significantly different from S. typhimurium control (P<.05). ***Significantly different from S. typhimurium control (P<.001). 2
4
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 9, 2015
io r
313
RESEARCH NOTE
To assess the level of colonization, four to nine chickens in each group were killed at 1,2, 4, 6, and 8 days after S. typhimurium inoculation, and cecal contents were removed for bacteriological examination. The number of 5. typhimurium in cecal contents of salmonella control and combined groups was counted and the number of test bacteria in cecal contents of the test control and combined groups was also counted. Bacteriological counts were replicated twice. Statistical analysis was conducted using Student's / test. RESULTS AND DISCUSSION 8
9
typhimurium were between 10 and 10 cfu/g from 1 day after inoculation to the end of the experiment (Figure la). These counts were compared with those of S. typhimurium in the combined groups. In the combined groups inoculated with B. vulgatus and S. typhimurium, B. thermophilum and S. typhimurium, or L. acidophilus and S. typhimurium, counts of S. typhimurium were 10 to 10 cfu/g for the entire experimental period with the exception of 1 day after S. typhimurium inoculation in the L. acidophilus combined group (Figure la,b,c,d). These counts were similar to those of the salmonella control group (Figure la). In the combined group inoculated with C. perfringens and S. typhimurium, the counts of S. typhimurium at 8 days after inoculation were significantly greater (P<.05; Figure 2e) than in the salmonella control group. The counts of each test bacteria were lower at 1 day after S. typhimurium inoculation than before S. typhimurium inoculation (Figure lb, c,e) with the exception of the L. acidophilus combined group (Figure Id). The counts of L. acidophilus at 1 day after S. typhimurium inoculation were the same as the counts of L. acidophilus in the test control group. At 2 days after S. typhimurium inoculation, the counts of L. acidophilus decreased (Figure Id). In the combined group inoculated with E. coli and 5. typhimurium, the counts of S. typhimurium (Figure If) were lower than in the salmonella control group (Figure la) through the entire experimental period. The counts of E. coli were not influenced by 5. typhimurium inoculation. The counts of test bacteria remained low for at least two sampling periods before returning to approximately the same
REFERENCES Barnes, E. M , C. S. Impey, and D. M. Cooper, 1980a. Competitive exclusion of salmonellas from the newly hatched chicken. Vet. Rec. 106:61. Barnes, E. M , C. S. Impey, and D. M. Cooper, 1980b. Manipulation of the crop and intestinal flora of the newly hatched chick. Am. J. Clin. Nutr. 33:2426-2433. Barnes, E. ML, C. S. Impey, and B.J.H. Stevens, 1979. Factors affecting the incidences and anti-salmonella activity of the anaerobic caecal flora of the young chick. J. Hyg. 82:263-283. Barrow, P. A., and J. F. Tucker, 1986. Inhibition of colonization of the chicken caecum with Salmonella typhimurium by pre-treatment with strains of Escherichia coli. J. Hyg. 96:161-169. Fukata, T., E. Baba, and A. Arakawa, 1987a. Invasion of Salmonella typhimurium into the cecal wall of gnotobiotic chickens with Eimeria tenella. Poultry Sci. 66:760-761. Fukata, T„ A. Kageyama, E. Baba, and A. Arakawa, 1987b. Effect of infection with Eimeria tenella upon the cecal bacterial population in monoflora chickens. Poultry Sci. 66:841-844. Hudault, S., H. Bewa, C. Bridonneau, and C. Raibaud, 1985. Efficiency of various bacterial suspension derived from ceca flora of conventional chickens in reducing the population level of Salmonella typhimurium in gnotobiotic mice and chicken intestines. Can. J. Microbiol. 31:832-838. Impey, C. S., G. C. Mead, and S. M. George, 1982. Competitive exclusion of salmonellas from the chick caecum using a defined mixture of bacterial isolated from the caecal microflora of an adult bird. J. Hyg. 89:479-490. Kageyama, A., T. Fukata, E. Baba, and A. Arakawa, 1987. The influence of various bacteria on the cecal mucosa on monoflora chickens infected with Eimeria tenella. A scanning electron microscopic study. Zentralbl. Baktcriol. Mikrobiol. Hyg. Ser. A 265:353-359. Lafont, J. P., A. Bree, M. Naciri, P. Yvore, J. F. Guillot, and
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 9, 2015
In the salmonella control, the counts of S.
level as 1 day after S. typhimurium inoculation. In general, after combined exposure to 5. typhimurium and test bacteria, either the population of S. typhimurium, but not the test bacteria was suppressed, or the population of test bacteria, but not 5. typhimurium, was suppressed. Thus B. vulgatus, C. perfringens, B. thermophilus, and L. acidophilus temporarily was suppressed by S. typhimurium populations. This suppression, though not significant, was consistent within the four test bacteria. However, E. coli was not suppressed by S. typhimurium at 1 and 6 days after inoculation, a pattern that continued throughout the experiment. In one instance, C. perfringens appeared to enhance populations of S. typhimurium after 8 days. Escherichia coli may play a major role in suppressing populations of S. typhimurium in the cecum of chickens.
314
FUKATA EI At.
E. Chaslus-Dancla, 1983. Experimental study of some factors limiting 'competitive exclusion' of salmonella in chickens. Res. Vet. Sci. 34:16-20. Nurmi, E., and M. Rantala, 1973. New aspects of Salmonella infection in broiler production. Nature, London 241:210-211. Rigby, C. E., J. R. Pettit, and A. Robertson, 1977. The effect of normal intestinal flora on the Salmonella carrier state in poultry with special reference to S. thompson and 5. typhimurium. Page 263 in: Proc. Int. Symp. on
Salmonella and Prospects for Control. D. A. Barnum, ed. University of Guelph. Guelph, Ontario, Canada. Soerjadi, A. S., A. B. Lloyd, and R. B. Cumming, 1978. Streptococcus faecalis, a bacterial isolate which protects young chickens from enteric invasion by salmonellae. Aust. Vet. J. 54:549-550. Watkins, B. A., and B. F. Miller, 1983. Competitive gut exclusion of avian pathogens by Lactobacillus acidophilus in gnotobiotic chicks. Poultry Sci. 62:1772-1779.
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 9, 2015