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Effect of Fructooligosaccharide on Salmonella Colonization of the Chicken Intestine
Effect of Fructooligosaccharide on Salmonella of the Chicken Intestine
Colonization
J. S. BAILEY, L. C. BLANKENSHIP, and N. A. COX USDA, Agricultural Research Service, Russell Research Center, Athens, Georgia 30613 (Received for publication May 15, 1991)
1991 Poultry Science 70:2433-2438 INTRODUCTION
Practical experience has demonstrated that it is difficult to reduce the percentage incidence of Salmonella on chickens to less than that already on the outside surface of chickens when they arrive at the processing plant. Significant reduction in Salmonella on processed broiler carcasses will therefore require the delivery of Salmonella-ieduced chickens to the processing plant. Preventing live chickens from becoming colonized with Salmonella during grow-out is extremely difficult because young chicks are highly susceptible to Salmonella colonization (Milner and Shaffer, 1952) and there are numerous potential sources of Salmonella. These include breeder stock (Cox et al., 1991), hatchery and hatchery environment (Cox et al., 1990), growout house and environment (Lahellec and Colin, 1985), and feed (Voeten et al, 1974; MacKenzie and Bains, 1976). Two potential approaches for control of Salmonella during grow-out are being tested. The concept of competitively excluding Salmonella from the gut of chickens was first introduced by Nurmi and Rantala (1973). Competitive exclusion (CE) was extensively reviewed by Pivnick and Nurmi (1982) and can best be summarized as follows: 1) newly hatched chicks may be colonized by a single
cell of Salmonella; 2) older birds are resistant to colonization because of established intestinal microflora; 3) the introduction of bacteria from an adult bird into a day-old chick speeds the maturation process of the gut microflora and increases the resistance of most chicks to Salmonella colonization. Several large-scale field studies have demonstrated varying degrees of CE effectiveness. The largest of these studies was that of Goren et al. (1988), whose study included eight million broilers. The study documented that CE treatment reduced Salmonella colonization by fourfold as compared with untreated controls. An alternative approach to control Salmonella during grow-out has been the addition of carbohydrates to the diet of chickens. Mannose or lactose in the diet of chickens has been reported to reduce Salmonella colonization (Oyofo et al., 1989a), but dextrose, maltose, or sucrose had no effect on the level of colonization. In vitro studies suggest that mannose may inhibit Salmonella colonization by blocking attachment sites in the gut (Oyofo et al., 1989b). However, McHan et al. (1989) found only minimal reduction in attachment of Salmonella with mannose. Lactose diets may lead to lower pH in the gut or changes in volatile fatty acids or both (Corrier et al., 1990a). Control of Salmonella colonization
2433
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ABSTRACT The influence of fructooligosaccharide (FOS) on the ability of Salmonella typhimurium to grow and colonize the gut of chickens was investigated. In vitro studies showed that Salmonella did not grow when FOS was the sole carbon source. When FOS was fed to chicks at the .375% level, little influence on Salmonella colonization was observed. At the .75% leveL 12% fewer FOS-fed birds were colonized with Salmonella compared with control birds. When chicks given a partially protective competitive exclusion (CE) culture were fed diets supplemented with .75% FOS, only 4 of 21 (19%) chickens challenged with 109 Salmonella cells on Day 7 became colonized as compared with 14 of 23 (61%) chickens given CE alone. When chickens were stressed by feed and water deprivation on Day 13 and challenged with 10 9 Salmonella on Day 14, 33 of 36 (92%) chickens fed a control diet were colonized compared with only 9 of 36 (25%) chickens fed a .75% FOS diet. Chickens treated with FOS had a fourfold reduction in the level of Salmonella present in the ceca. Feeding FOS in the diet of chickens may lead to a shift in the intestinal gut microflora, and under some circumstances may result in reduced susceptibility to Salmonella colonization. (Key words: Salmonella, fructooligosaccharide, chicken, colonization, competitive exclusion)
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BAILEY ET AL.
MATERIALS AND METHODS
In Vitro Study Twenty different serotypes (Table 1) of Salmonella that had previously been maintained on double-strength nutrient agar stabs were
'CoorsBioTech, Westminster, CO 80234. Milton Roy, Rochester, NY 14625. ^ o w Laboratories, McLean, VA 22102. kernel, Lenexa, KS 66215. 2
streaked onto brain heart infusion (BHT) agar slants and incubated for 20 ± 2 h at 35 C. Cultures were washed from the slants with sterile physiological saline and serially diluted to an optical density of .2 as measured by a Spectronic 710 spectrophotometer2 using a wavelength of 540 nm. At this concentration there are approximately 108 cells of Salmonella per milliliter. Cultures were further serially diluted in physiological saline so that each 10 |iL contained about 200 cells of Salmonella. Minimal media (Gomez et al., 1973) was dispensed (200 uL per well) into microliter plates in duplicate with either 0, .25, .5, 1.0, or 2.0% FOS or glucose added. Approximately 200 cells of the appropriate Salmonella were aseptically placed in each well. Plates with inoculum were placed in a container with a moist towel to prevent drying and incubated for 18 to 20 h at 35 C. The optical density of the cultures was then determined at 405 nm with a Titertek Multiskan Plus.3 The entire experiment was replicated once. Competitive Exclusion Culture The CE culture was prepared by aseptically removing about 1 g of ceca with contents from a 25-wk-old, Salmonella-free laying chicken, immediately placing it into a 5-mL tube of prereduced anaerobic (PRAS) BHI 4 and incubated for 48 h at 35 C. Fifteen percent glycerol was added to the incubated culture, which was then split into .5-mL aliquots and stored in a-80 C freezer until use. Two days prior to treatment of chicks, a tube of CE was thawed and .2 mL of the culture added to a new 5 mL of PRAS BHI tube which was incubated at 35 C for 48 h. In Vivo: Fructooligosaccharide in Drinking Water Broiler chicks were obtained from a local hatchery on the day of hatch and transported to the laboratory. Six chicks were randomly placed into 14 isolator units at the University of Georgia Poultry Disease Research Center. Chicks were provided ad libitum access to an unmedicated, broiler starter crumble feed that met or exceeded the National Research Council requirements (1984) and given either plain water or water with 2% FOS for the duration of the experiment. One group of chicks was orally gavaged with .2 mL of a 48-h CE culture. All chicks were challenged orally by gavage on Day
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with mannose or lactose has shown mixed results (Oyofo et al., 1989a; Corner et al., 1990b; Hinton et al., 1990), and most effective results have come from the combination of CE with lactose (Hinton et al, 1990). Whether through addition of CE cultures, natural maturation of intestinal bacteria, or changes in gut populations caused by reduction in pH, the intestinal microflora of chickens has an important influence on gut colonization by microorganisms, including Salmonella. The intestinal microflora is also quantitatively and qualitatively influenced by the composition of the diet. Thus, a potential exists to manipulate the diet of chickens with dietary amendments that would favor development of a microflora that produces a hostile environment for enteropathogens and thereby, prevent their colonization in the gut. Fructooligosaccharides1 (FOS) reportedly can be substituted for subtherapeutic levels of antibiotics to enhance the growth and production efficiency of broilers (Ammerman et al., 1988a,b, 1989). Fructooligosaccharide consists primarily of one to three fructose residues attached to a sucrose molecule and is found naturally in relatively high concentrations in onions, wheat, barley, and rye. Fructooligosaccharide has been shown to influence intestinal bacterial populations by enhancing the growth of Lactobacillus species (Mitsuoka et al., 1987) and Bifidobacterium (Hidaka et al., 1986). Therefore, the objectives of the present study were 1) to determine the effect of FOS on in vitro growth of Salmonella when FOS is the sole carbon source; and 2) to determine the effect on Salmonella colonization of chickens fed FOS alone, FOS in combination with CE, and an FOS diet followed by feed withdrawal stress.
CONTROL OF SALMONELLA
2435
TABLE 1. Salmonella serotypes tested for ability to utilize fructooligosaccharide as the sole carbon source S. S. S. S. S.
gallinarium California mission westhampton Chester
S. S. S. S. S.
meleagridis heidelberg senftenberg siegburg amager
S. S. S. S. S.
S. S. S. S. S.
orion wentworth thompson cerro bredeney
menhaden taksony drypool florida typhimurium
feed and water for 24 h. Stressed and unstressed chicks were then challenged by oral gavage with 109 cells of the marker strain of S. typhimurium and returned to their pens. On Day 20, half of the chicks in each treatment group in each pen were placed in coops to stimulate commercial feed and water withdrawal practices. The other half of the chicks were kept on feed and water until they were caught and killed. All chicks were killed on Day 21 and the ceca examined semiquantitatively for the presence of marker S. typhimurium. This experiment was replicated once.
In Vivo: Fructooligosaccharide in Feed
Statistical Analysis
Broiler chicks were obtained and managed similarly to the FOS in water trial. Chicks were provided ad libitum access to unmedicated, broiler starter crumble feed with either 0, .375, or .75% added FOS for the duration of the trials. Seven days after placement, chicks were orally challenged with either 106, 108, or 109 cells of the strain of S. typhimurium resistant to nalidixic acid. On Day 14, the chicks were killed and analyzed for the marker Salmonella semiquantitatively, as described above. One trial was run with the .375% added FOS and two trials were run with the .75% added FOS. In addition, chicks were orally treated with CE as above and fed either control feed or FOS-supplemented feed. In Vivo: Fructooligosaccharide and Stress Broiler chicks were picked up from a local hatchery on the day of hatch and transported to the University of Georgia Poultry Farm. Chicks were held in floor pens and were provided ad libitum access to either an unmedicated broiler starter crumble feed or the same feed with .75% FOS for the duration of the trials. On Day 13, half of these chicks were stressed by removing
Treatments were tested using the General Linear Models (GLM) procedure of SAS® software (SAS Institute, 1985). Orthogonal contrasts were used to compare means. Percentage positives were analyzed after arc sine transformations. Significance was defined as P<.05. RESULTS AND DISCUSSION
In Vitro Study The mean optical density reading for the Salmonella was greater than .35 in the glucose media and less than .05 for the FOS media (data not shown). None of the 20 serotypes of Salmonella tested differed significantly from these figures, indicating that Salmonella cannot grow when FOS is the only carbon source. In Vivo: Fructooligosaccharide in Water Chicks administered 2% FOS in the water and challenged with Salmonella on Day 2 had low levels of detectable Salmonella and no statistical differences from the control were observed (Table 2). The best overall reduction in Salmonella colonization was seen with the CE treatment. The effectiveness of CE in these trials
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2 with 106 cells or on Day 7 with 106 or 108 cells of a strain of Salmonella typhimurium resistant to nalidixic acid (Bailey et al., 1988). On Day 14, chicks were killed by cervical dislocation and presence of the marker S. typhimurium was semiquantitatively determined (Bailey et al., 1988). Results were recorded quantitatively as the number of Salmonella-positive chicks per number of chicks in the treatment group, and qualitatively as the colonization factor (CF) or mean number of Salmonella per gram of ceca for all birds within a treatment group. This experiment was replicated once.
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BAILEY ET AL.
TABLE 2. Effect of water-administered fructooligosaccharide (FOS) and competitive exclusion (CE) on the colonization of young chicks with Salmonella Challenge Treatment
Day
1 2 1 2 1 2 1 2 1 2 1 2 1 2
Level 6
10 106 10 6 106 106 106 10 6 106 108 108 10 6 10 6 108 108
Sa/mone/Za-positive/ chicks tested 6/6 5/6 2/6 5/6 0/6 2/6 0/6 2/6 0/6 3/6 0/6 0/6 0/6 3/4
CF 1 1.9 2.8 .7 2.0 .5 .5 3.0
2.2
CF (colonization factor) = mean log Salmonella count per gram of ceca for all birds within a treatment group.
was similar to that observed previously (Nurmi and Rantala, 1973; Bailey et al, 1988; Goren et al., 1988). When chicks were not challenged until 7 days, the level of colonization in the control chicks was low, making it difficult to evaluate the effectiveness of the FOS. The difficulty in colonizing older chicks has been observed in the authors' and other laboratories before, and is probably attributable to the development of a natural gut flora. If FOS were used in field situations, the probable method of application would not be in the water, but rather in the feed. Therefore, in all subsequent experiments the FOS was administered in the feed. In Vivo: Fructooligosaccharide in Feed When FOS was fed continuously at the .375% level, there was little difference in the level of Salmonella colonization between control and FOS-fed chicks. In two subsequent trials (Table 3), the level of FOS was increased in the feed to .75% in an attempt to maximize possible effects on Salmonella colonization. When treatment means were compared by orthogonal contrasts using the treatment by level interaction as the error term, FOS treatment alone did not differ significantly from the other treatments, but the CE plus FOS combination treatment was significantly different from all of the other treatments. The FOS-fed birds had only about
12% less Salmonella colonization, with a resulting reduction in the CF of .75 log Salmonella when compared with control birds. Chicks given CE on the day of hatch had a 24% reduction in Salmonella-positive colonization and greater than a 2 log reduction in the CF. The CE culture used in the present trial had been maintained in a 15% glycerol solution in a -80 C freezer for about 2 mo and may have lost some efficacy. Loss of efficacy over time of storage had been previously reported (Bailey et al., 1988). With the CE culture giving less than complete protection, the least amount of Salmonella colonization was observed when the CE was used in combination with continuous feeding of FOS. With this combination, only 4 of 21 chicks challenged with 109 Salmonella were colonized, with only a CF of log .5 Salmonella/g of ceca as compared with controls among which 21 of 22 untreated chicks were colonized and 14 of 23 with CE treatment alone. These results are similar to Hinton et al. (1990), who found that chicks continuously given water containing 2.5% lactose and given a less than effective CE on the day of hatch had better protection from colonization than those given the CE alone or the lactose water alone. In Vivo: Fructooligosaccharide and Stress When treatment means were compared by orthogonal contrasts using the diet by stress by
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None None 2% FOS 2% FOS CE Day 0 CE Day 0 None None None None 2% FOS 2% FOS 2% FOS 2% FOS
Replication
2437
CONTROL OF SALMONELLA
TABLE 3. Effect of .75% fructooligosaccharide (FOS) in the feed and administration of competitive exclusion (CE) on the Salmonella colonization of 7-day-old chicks
Treatment
6
1 2 1 2 1 2 1 2 1 2 1 2 FOS FOS FOS FOS
6/6 4/15 7/7 14/15 7/7 1/15 8/8 12/15 8/8 2/15 8/8 6/15 0/4 2/15 2/6 2/15
10 10 6 109 10 9 106 106 109 109 10 6 10 6 109 109 10 6 10 6 109 109
1 2 1 2
Salmonella-positive/ chicks tested
CF 1 7.7 .4 6.3 3.0 6.6 .1 5.7 2.1 1.7 .2 2.0 .9 .2 .7 .4
X
CF (colonization factor) = mean log Salmonella count per gram of ceca for all birds within a treatment group.
replication interaction as the error term, there were no differences in the number of Salmonella-positive birds between control and FOS diets; however, when the birds were stressed, significantly more Salmonella-positive birds were found in the control group than in the group given the FOS diet. Chicks given feed with no added FOS or feed with .75% FOS each had 4 of 36 (11%) chicks colonized with Salmonella at Day 21 when they were challenged with 109 Salmonella on Day 14 (Table 4). When an equal group of chicks was deprived of feed and water on Day 13 before challenge with Salmonella on Day 14, 33 of the 36 (92%) chicks not given FOS were colonized, but only 9 of 36 (25%) chicks on feed with .75% FOS were colonized.
A possible explanation for these differences is that the gut microbial population of the chickens fed an FOS diet was changed from the control chickens, and that the altered gut microflora was not as susceptible to the stress of feed and water deprivation as the microflora of birds provided the control diet. The possible changes in the microbial population were not specifically identified in the current study. In an earlier clinical study with humans (Hidaka et al., 1986), FOS in the diet led to an increased number of Bifidobacteria with subsequent relief of constipation, improved blood lipids in hyperlipidemia, and a suppressed production of intestinal putrefactive substances.
TABLE 4. Effect of .75% fructooligosaccharide (FOS) in the feed on the Salmonella colonization of 14-day-old chicks stressed by feed and water deprivation
Diet
Treatment
Replication
Control Control Control Control FOS FOS FOS FOS
Unstressed Unstressed Stressed Stressed Unstressed Unstressed Stressed Stressed
1 2 1 2 1 2 1 2
Challenge level
Salmonella--positive/ chicks tested
109 109 109 109
3/12 1/24 12/12 21/24
109 109 109 109
4/12 0/24 6/12 3/24
CF 1 .5 .1 2.6 1.9 .5 .8 .2
CF (colonization factor) = mean log Salmonella count per gram of ceca for all birds within a treatment group.
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None None None None FOS FOS FOS FOS CE CE CE CE CE + CE + CE + CE +
Challenge level
Replication
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BAILEY ET AL.
In summary, Salmonella cannot grow in vitro when FOS is the sole carbon source. When FOS was fed at levels of .375 or .75%, little influence on Salmonella colonization was observed, but when the FOS was fed in combination with a CE treatment, there was an observed reduction in both the number of Salmonella-positive birds and the number of Salmonella per gram of ceca. When birds were stressed by feed and water deprivation, groups consuming a .75% FOS diet had both fewer Salmonella-positive birds and about fourfold fewer Salmonella per gram of ceca than did controls.
The authors thank Debbie Posey and Lalla Tanner for their able technical assistance in the completion of this study. REFERENCES Ammennan, E., C. Quarles, and P. V. Twining, Jr., 1988a. Effect of dietary fructooligosaccharides on feed efficiency in floor-pen reared male broilers. Poultry Sci. 67(Suppl. l):l.(Abstr.) Ammennan, E., C. Quarles, and P. V. Twining, Jr., 1988b. Broiler response to the addition of dietary fructooligosaccharides. Poultry Sci. 67(Suppl. 1): 46.(Abstr.) Ammennan, E., C. Quarles, and P. V. Twining, Jr., 1989. Evaluation of fructooligosaccharides on performance and carcass yield of male broilers. Poultry Sci. 68(Suppl. l):167.(Abstr.) Bailey, J. S., L. C. Blankenship, N. J. Stern, N. A. Cox, and F. McHan, 1988. Effect of anticoccidial and antimicrobial feed additives on prevention of Salmonella colonization of chicks treated with anaerobic cultures of chicken feces. Avian Dis. 32:324-329. Corrier, D. E., A. Hinton, Jr., R. L. Ziprin, R. C. Beier, and J. R. DeLoach, 1990a. Effect of dietary lactose on cecal pH, bacteriostatic volatile fatty acids, and Salmonella typhimurium colonization of broiler chicks. Avian Dis. 34:617-625. Corner, D. E., A. Hinton, Jr., R. L. Ziprin, and J. R. DeLoach, 1990b. Effect of dietary lactose on Salmonella colonization of market-age broiler chickens. Avian Dis. 34:668-676. Cox, N. A., J. S. Bailey, J. M. Mauldin, and L. C. Blankenship, 1990a. Presence and impact of Salmonella contamination in commercial broiler hatcheries. Poultry Sci. 69:1606-1609. Cox, N. A., J. S. Bailey, J. M. Mauldin, L. C. Blankenship, and J. L. Wilson, 1991. Extent of salmonellae contamination in breeder hatcheries. Poultry Sci. 70: 416-418.
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ACKNOWLEDGMENTS
Gomez, R. F., A. J. Sinskey, R. Davies, and T. P. Labuza, 1973. Minimal media for the isolation of heated Salmonella typhimurium LT2. Gen. Microbiol. 74: 267-274. Goren, E., W. A. de Jong, P. Doornenbal, N. M. Bolder, R.W.A.W. Mulder, and J. Jansen, 1988. Reduction of salmonellae infection of broilers by spray application of intestinal microflora: a longitudinal study. Vet. Q. 10:249-255. Hinton, J., Jr., D. E. Corner, G. E. Spates, J. O. Norman, R. L. Zipprin, R. C. Beier, and J. R. DeLoach, 1990. Biological control of Salmonella typhimurium in young chickens. Avian Dis. 34:626-633. Hidaka, H., T. Edida, T. Takizawa, T. Tokunaga, and Y. Tashiro, 1986. Effects of fructooligosaccharides on intestinal flora and human health. Bifidobacteria Microflora 5:37-50. Lahellec, C, and P. Colin, 1985. Relationship between serotypes of Salmonella from hatcheries and rearing farms and those from processed poultry carcasses. Br. Poult. Sci. 26:179-186. MacKenzie, M. A., and B. S. Bains, 1976. Dissemination of Salmonella serotypes from raw feed ingredients to chicken carcasses. Poultry Sci. 55:957-960. McHan, F., N. A. Cox, L. C. Blankenship, and J. S. Bailey, 1989. In vitro attachment of Salmonella typhimurium to chick ceca exposed to selected carbohydrates. Avian Dis. 33:340-344. Milner, K. C , and M. F. Shaffer, 1952. Bacteriologic studies of experimental Salmonella infections in chicks. J. Infect. Dis. 90:81-85. Mitsuoka, T., H. Hidaka, and T. Eida, 1987. Effect of fructooligosaccharides on intestinal microflora. Die Nahrung 31:5-6, 427-436. National Research Council, 1984. Nutrient Requirements of Poultry. 8th rev. ed. National Academy Press, Washington, DC. Nurmi, E., and M. Rantala, 1973. New aspects of Salmonella infection in broiler production. Nature 214:210-211. Oyofo, B. A., J. R. DeLoach, D. E. Corrier, J. O. Norman, R. L. Ziprin, and H. H. MoUenuauer, 1989a. Effect of carbohydrates on Salmonella typhimurium colonization in broiler chickens. Avian Dis. 33:531-534. Oyofo, B. A., R. E. Droleskey, J. O. Norman, H. H. Mollenhauer, R. L. Ziprin, D. E. Corrier, and J. R. DeLoach. 1989b. Inhibition by mannose of in vitro colonization of chicken small intestine by Salmonella typhimurium. Poultry Sci. 68:1351-1356. Pivnick, H., and E. Nurmi, 1982. The Nurmi concept and its role in the control of salmonellae in poultry. Page 41 in: Developments in Food Microbiology Volume 1, Chapter 2, R. Davis, ed. Applied Science, Barking, UK. SAS Institute, 1985. SAS® User's Guide: Statistics. Version 5 Edition. SAS Institute Inc., Cary, NC. Voeten, A. C , DJH.J. Bros, and FJIJ. Jaartsveld, 1974. Distribution of Salmonella within a closed broiler chicken operation. Tydsk. Diergeneeskd. 99: 1093-1094.
Colonization
J. S. BAILEY, L. C. BLANKENSHIP, and N. A. COX USDA, Agricultural Research Service, Russell Research Center, Athens, Georgia 30613 (Received for publication May 15, 1991)
1991 Poultry Science 70:2433-2438 INTRODUCTION
Practical experience has demonstrated that it is difficult to reduce the percentage incidence of Salmonella on chickens to less than that already on the outside surface of chickens when they arrive at the processing plant. Significant reduction in Salmonella on processed broiler carcasses will therefore require the delivery of Salmonella-ieduced chickens to the processing plant. Preventing live chickens from becoming colonized with Salmonella during grow-out is extremely difficult because young chicks are highly susceptible to Salmonella colonization (Milner and Shaffer, 1952) and there are numerous potential sources of Salmonella. These include breeder stock (Cox et al., 1991), hatchery and hatchery environment (Cox et al., 1990), growout house and environment (Lahellec and Colin, 1985), and feed (Voeten et al, 1974; MacKenzie and Bains, 1976). Two potential approaches for control of Salmonella during grow-out are being tested. The concept of competitively excluding Salmonella from the gut of chickens was first introduced by Nurmi and Rantala (1973). Competitive exclusion (CE) was extensively reviewed by Pivnick and Nurmi (1982) and can best be summarized as follows: 1) newly hatched chicks may be colonized by a single
cell of Salmonella; 2) older birds are resistant to colonization because of established intestinal microflora; 3) the introduction of bacteria from an adult bird into a day-old chick speeds the maturation process of the gut microflora and increases the resistance of most chicks to Salmonella colonization. Several large-scale field studies have demonstrated varying degrees of CE effectiveness. The largest of these studies was that of Goren et al. (1988), whose study included eight million broilers. The study documented that CE treatment reduced Salmonella colonization by fourfold as compared with untreated controls. An alternative approach to control Salmonella during grow-out has been the addition of carbohydrates to the diet of chickens. Mannose or lactose in the diet of chickens has been reported to reduce Salmonella colonization (Oyofo et al., 1989a), but dextrose, maltose, or sucrose had no effect on the level of colonization. In vitro studies suggest that mannose may inhibit Salmonella colonization by blocking attachment sites in the gut (Oyofo et al., 1989b). However, McHan et al. (1989) found only minimal reduction in attachment of Salmonella with mannose. Lactose diets may lead to lower pH in the gut or changes in volatile fatty acids or both (Corrier et al., 1990a). Control of Salmonella colonization
2433
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ABSTRACT The influence of fructooligosaccharide (FOS) on the ability of Salmonella typhimurium to grow and colonize the gut of chickens was investigated. In vitro studies showed that Salmonella did not grow when FOS was the sole carbon source. When FOS was fed to chicks at the .375% level, little influence on Salmonella colonization was observed. At the .75% leveL 12% fewer FOS-fed birds were colonized with Salmonella compared with control birds. When chicks given a partially protective competitive exclusion (CE) culture were fed diets supplemented with .75% FOS, only 4 of 21 (19%) chickens challenged with 109 Salmonella cells on Day 7 became colonized as compared with 14 of 23 (61%) chickens given CE alone. When chickens were stressed by feed and water deprivation on Day 13 and challenged with 10 9 Salmonella on Day 14, 33 of 36 (92%) chickens fed a control diet were colonized compared with only 9 of 36 (25%) chickens fed a .75% FOS diet. Chickens treated with FOS had a fourfold reduction in the level of Salmonella present in the ceca. Feeding FOS in the diet of chickens may lead to a shift in the intestinal gut microflora, and under some circumstances may result in reduced susceptibility to Salmonella colonization. (Key words: Salmonella, fructooligosaccharide, chicken, colonization, competitive exclusion)
2434
BAILEY ET AL.
MATERIALS AND METHODS
In Vitro Study Twenty different serotypes (Table 1) of Salmonella that had previously been maintained on double-strength nutrient agar stabs were
'CoorsBioTech, Westminster, CO 80234. Milton Roy, Rochester, NY 14625. ^ o w Laboratories, McLean, VA 22102. kernel, Lenexa, KS 66215. 2
streaked onto brain heart infusion (BHT) agar slants and incubated for 20 ± 2 h at 35 C. Cultures were washed from the slants with sterile physiological saline and serially diluted to an optical density of .2 as measured by a Spectronic 710 spectrophotometer2 using a wavelength of 540 nm. At this concentration there are approximately 108 cells of Salmonella per milliliter. Cultures were further serially diluted in physiological saline so that each 10 |iL contained about 200 cells of Salmonella. Minimal media (Gomez et al., 1973) was dispensed (200 uL per well) into microliter plates in duplicate with either 0, .25, .5, 1.0, or 2.0% FOS or glucose added. Approximately 200 cells of the appropriate Salmonella were aseptically placed in each well. Plates with inoculum were placed in a container with a moist towel to prevent drying and incubated for 18 to 20 h at 35 C. The optical density of the cultures was then determined at 405 nm with a Titertek Multiskan Plus.3 The entire experiment was replicated once. Competitive Exclusion Culture The CE culture was prepared by aseptically removing about 1 g of ceca with contents from a 25-wk-old, Salmonella-free laying chicken, immediately placing it into a 5-mL tube of prereduced anaerobic (PRAS) BHI 4 and incubated for 48 h at 35 C. Fifteen percent glycerol was added to the incubated culture, which was then split into .5-mL aliquots and stored in a-80 C freezer until use. Two days prior to treatment of chicks, a tube of CE was thawed and .2 mL of the culture added to a new 5 mL of PRAS BHI tube which was incubated at 35 C for 48 h. In Vivo: Fructooligosaccharide in Drinking Water Broiler chicks were obtained from a local hatchery on the day of hatch and transported to the laboratory. Six chicks were randomly placed into 14 isolator units at the University of Georgia Poultry Disease Research Center. Chicks were provided ad libitum access to an unmedicated, broiler starter crumble feed that met or exceeded the National Research Council requirements (1984) and given either plain water or water with 2% FOS for the duration of the experiment. One group of chicks was orally gavaged with .2 mL of a 48-h CE culture. All chicks were challenged orally by gavage on Day
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on May 24, 2015
with mannose or lactose has shown mixed results (Oyofo et al., 1989a; Corner et al., 1990b; Hinton et al., 1990), and most effective results have come from the combination of CE with lactose (Hinton et al, 1990). Whether through addition of CE cultures, natural maturation of intestinal bacteria, or changes in gut populations caused by reduction in pH, the intestinal microflora of chickens has an important influence on gut colonization by microorganisms, including Salmonella. The intestinal microflora is also quantitatively and qualitatively influenced by the composition of the diet. Thus, a potential exists to manipulate the diet of chickens with dietary amendments that would favor development of a microflora that produces a hostile environment for enteropathogens and thereby, prevent their colonization in the gut. Fructooligosaccharides1 (FOS) reportedly can be substituted for subtherapeutic levels of antibiotics to enhance the growth and production efficiency of broilers (Ammerman et al., 1988a,b, 1989). Fructooligosaccharide consists primarily of one to three fructose residues attached to a sucrose molecule and is found naturally in relatively high concentrations in onions, wheat, barley, and rye. Fructooligosaccharide has been shown to influence intestinal bacterial populations by enhancing the growth of Lactobacillus species (Mitsuoka et al., 1987) and Bifidobacterium (Hidaka et al., 1986). Therefore, the objectives of the present study were 1) to determine the effect of FOS on in vitro growth of Salmonella when FOS is the sole carbon source; and 2) to determine the effect on Salmonella colonization of chickens fed FOS alone, FOS in combination with CE, and an FOS diet followed by feed withdrawal stress.
CONTROL OF SALMONELLA
2435
TABLE 1. Salmonella serotypes tested for ability to utilize fructooligosaccharide as the sole carbon source S. S. S. S. S.
gallinarium California mission westhampton Chester
S. S. S. S. S.
meleagridis heidelberg senftenberg siegburg amager
S. S. S. S. S.
S. S. S. S. S.
orion wentworth thompson cerro bredeney
menhaden taksony drypool florida typhimurium
feed and water for 24 h. Stressed and unstressed chicks were then challenged by oral gavage with 109 cells of the marker strain of S. typhimurium and returned to their pens. On Day 20, half of the chicks in each treatment group in each pen were placed in coops to stimulate commercial feed and water withdrawal practices. The other half of the chicks were kept on feed and water until they were caught and killed. All chicks were killed on Day 21 and the ceca examined semiquantitatively for the presence of marker S. typhimurium. This experiment was replicated once.
In Vivo: Fructooligosaccharide in Feed
Statistical Analysis
Broiler chicks were obtained and managed similarly to the FOS in water trial. Chicks were provided ad libitum access to unmedicated, broiler starter crumble feed with either 0, .375, or .75% added FOS for the duration of the trials. Seven days after placement, chicks were orally challenged with either 106, 108, or 109 cells of the strain of S. typhimurium resistant to nalidixic acid. On Day 14, the chicks were killed and analyzed for the marker Salmonella semiquantitatively, as described above. One trial was run with the .375% added FOS and two trials were run with the .75% added FOS. In addition, chicks were orally treated with CE as above and fed either control feed or FOS-supplemented feed. In Vivo: Fructooligosaccharide and Stress Broiler chicks were picked up from a local hatchery on the day of hatch and transported to the University of Georgia Poultry Farm. Chicks were held in floor pens and were provided ad libitum access to either an unmedicated broiler starter crumble feed or the same feed with .75% FOS for the duration of the trials. On Day 13, half of these chicks were stressed by removing
Treatments were tested using the General Linear Models (GLM) procedure of SAS® software (SAS Institute, 1985). Orthogonal contrasts were used to compare means. Percentage positives were analyzed after arc sine transformations. Significance was defined as P<.05. RESULTS AND DISCUSSION
In Vitro Study The mean optical density reading for the Salmonella was greater than .35 in the glucose media and less than .05 for the FOS media (data not shown). None of the 20 serotypes of Salmonella tested differed significantly from these figures, indicating that Salmonella cannot grow when FOS is the only carbon source. In Vivo: Fructooligosaccharide in Water Chicks administered 2% FOS in the water and challenged with Salmonella on Day 2 had low levels of detectable Salmonella and no statistical differences from the control were observed (Table 2). The best overall reduction in Salmonella colonization was seen with the CE treatment. The effectiveness of CE in these trials
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2 with 106 cells or on Day 7 with 106 or 108 cells of a strain of Salmonella typhimurium resistant to nalidixic acid (Bailey et al., 1988). On Day 14, chicks were killed by cervical dislocation and presence of the marker S. typhimurium was semiquantitatively determined (Bailey et al., 1988). Results were recorded quantitatively as the number of Salmonella-positive chicks per number of chicks in the treatment group, and qualitatively as the colonization factor (CF) or mean number of Salmonella per gram of ceca for all birds within a treatment group. This experiment was replicated once.
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BAILEY ET AL.
TABLE 2. Effect of water-administered fructooligosaccharide (FOS) and competitive exclusion (CE) on the colonization of young chicks with Salmonella Challenge Treatment
Day
1 2 1 2 1 2 1 2 1 2 1 2 1 2
Level 6
10 106 10 6 106 106 106 10 6 106 108 108 10 6 10 6 108 108
Sa/mone/Za-positive/ chicks tested 6/6 5/6 2/6 5/6 0/6 2/6 0/6 2/6 0/6 3/6 0/6 0/6 0/6 3/4
CF 1 1.9 2.8 .7 2.0 .5 .5 3.0
2.2
CF (colonization factor) = mean log Salmonella count per gram of ceca for all birds within a treatment group.
was similar to that observed previously (Nurmi and Rantala, 1973; Bailey et al, 1988; Goren et al., 1988). When chicks were not challenged until 7 days, the level of colonization in the control chicks was low, making it difficult to evaluate the effectiveness of the FOS. The difficulty in colonizing older chicks has been observed in the authors' and other laboratories before, and is probably attributable to the development of a natural gut flora. If FOS were used in field situations, the probable method of application would not be in the water, but rather in the feed. Therefore, in all subsequent experiments the FOS was administered in the feed. In Vivo: Fructooligosaccharide in Feed When FOS was fed continuously at the .375% level, there was little difference in the level of Salmonella colonization between control and FOS-fed chicks. In two subsequent trials (Table 3), the level of FOS was increased in the feed to .75% in an attempt to maximize possible effects on Salmonella colonization. When treatment means were compared by orthogonal contrasts using the treatment by level interaction as the error term, FOS treatment alone did not differ significantly from the other treatments, but the CE plus FOS combination treatment was significantly different from all of the other treatments. The FOS-fed birds had only about
12% less Salmonella colonization, with a resulting reduction in the CF of .75 log Salmonella when compared with control birds. Chicks given CE on the day of hatch had a 24% reduction in Salmonella-positive colonization and greater than a 2 log reduction in the CF. The CE culture used in the present trial had been maintained in a 15% glycerol solution in a -80 C freezer for about 2 mo and may have lost some efficacy. Loss of efficacy over time of storage had been previously reported (Bailey et al., 1988). With the CE culture giving less than complete protection, the least amount of Salmonella colonization was observed when the CE was used in combination with continuous feeding of FOS. With this combination, only 4 of 21 chicks challenged with 109 Salmonella were colonized, with only a CF of log .5 Salmonella/g of ceca as compared with controls among which 21 of 22 untreated chicks were colonized and 14 of 23 with CE treatment alone. These results are similar to Hinton et al. (1990), who found that chicks continuously given water containing 2.5% lactose and given a less than effective CE on the day of hatch had better protection from colonization than those given the CE alone or the lactose water alone. In Vivo: Fructooligosaccharide and Stress When treatment means were compared by orthogonal contrasts using the diet by stress by
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None None 2% FOS 2% FOS CE Day 0 CE Day 0 None None None None 2% FOS 2% FOS 2% FOS 2% FOS
Replication
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CONTROL OF SALMONELLA
TABLE 3. Effect of .75% fructooligosaccharide (FOS) in the feed and administration of competitive exclusion (CE) on the Salmonella colonization of 7-day-old chicks
Treatment
6
1 2 1 2 1 2 1 2 1 2 1 2 FOS FOS FOS FOS
6/6 4/15 7/7 14/15 7/7 1/15 8/8 12/15 8/8 2/15 8/8 6/15 0/4 2/15 2/6 2/15
10 10 6 109 10 9 106 106 109 109 10 6 10 6 109 109 10 6 10 6 109 109
1 2 1 2
Salmonella-positive/ chicks tested
CF 1 7.7 .4 6.3 3.0 6.6 .1 5.7 2.1 1.7 .2 2.0 .9 .2 .7 .4
X
CF (colonization factor) = mean log Salmonella count per gram of ceca for all birds within a treatment group.
replication interaction as the error term, there were no differences in the number of Salmonella-positive birds between control and FOS diets; however, when the birds were stressed, significantly more Salmonella-positive birds were found in the control group than in the group given the FOS diet. Chicks given feed with no added FOS or feed with .75% FOS each had 4 of 36 (11%) chicks colonized with Salmonella at Day 21 when they were challenged with 109 Salmonella on Day 14 (Table 4). When an equal group of chicks was deprived of feed and water on Day 13 before challenge with Salmonella on Day 14, 33 of the 36 (92%) chicks not given FOS were colonized, but only 9 of 36 (25%) chicks on feed with .75% FOS were colonized.
A possible explanation for these differences is that the gut microbial population of the chickens fed an FOS diet was changed from the control chickens, and that the altered gut microflora was not as susceptible to the stress of feed and water deprivation as the microflora of birds provided the control diet. The possible changes in the microbial population were not specifically identified in the current study. In an earlier clinical study with humans (Hidaka et al., 1986), FOS in the diet led to an increased number of Bifidobacteria with subsequent relief of constipation, improved blood lipids in hyperlipidemia, and a suppressed production of intestinal putrefactive substances.
TABLE 4. Effect of .75% fructooligosaccharide (FOS) in the feed on the Salmonella colonization of 14-day-old chicks stressed by feed and water deprivation
Diet
Treatment
Replication
Control Control Control Control FOS FOS FOS FOS
Unstressed Unstressed Stressed Stressed Unstressed Unstressed Stressed Stressed
1 2 1 2 1 2 1 2
Challenge level
Salmonella--positive/ chicks tested
109 109 109 109
3/12 1/24 12/12 21/24
109 109 109 109
4/12 0/24 6/12 3/24
CF 1 .5 .1 2.6 1.9 .5 .8 .2
CF (colonization factor) = mean log Salmonella count per gram of ceca for all birds within a treatment group.
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None None None None FOS FOS FOS FOS CE CE CE CE CE + CE + CE + CE +
Challenge level
Replication
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BAILEY ET AL.
In summary, Salmonella cannot grow in vitro when FOS is the sole carbon source. When FOS was fed at levels of .375 or .75%, little influence on Salmonella colonization was observed, but when the FOS was fed in combination with a CE treatment, there was an observed reduction in both the number of Salmonella-positive birds and the number of Salmonella per gram of ceca. When birds were stressed by feed and water deprivation, groups consuming a .75% FOS diet had both fewer Salmonella-positive birds and about fourfold fewer Salmonella per gram of ceca than did controls.
The authors thank Debbie Posey and Lalla Tanner for their able technical assistance in the completion of this study. REFERENCES Ammennan, E., C. Quarles, and P. V. Twining, Jr., 1988a. Effect of dietary fructooligosaccharides on feed efficiency in floor-pen reared male broilers. Poultry Sci. 67(Suppl. l):l.(Abstr.) Ammennan, E., C. Quarles, and P. V. Twining, Jr., 1988b. Broiler response to the addition of dietary fructooligosaccharides. Poultry Sci. 67(Suppl. 1): 46.(Abstr.) Ammennan, E., C. Quarles, and P. V. Twining, Jr., 1989. Evaluation of fructooligosaccharides on performance and carcass yield of male broilers. Poultry Sci. 68(Suppl. l):167.(Abstr.) Bailey, J. S., L. C. Blankenship, N. J. Stern, N. A. Cox, and F. McHan, 1988. Effect of anticoccidial and antimicrobial feed additives on prevention of Salmonella colonization of chicks treated with anaerobic cultures of chicken feces. Avian Dis. 32:324-329. Corrier, D. E., A. Hinton, Jr., R. L. Ziprin, R. C. Beier, and J. R. DeLoach, 1990a. Effect of dietary lactose on cecal pH, bacteriostatic volatile fatty acids, and Salmonella typhimurium colonization of broiler chicks. Avian Dis. 34:617-625. Corner, D. E., A. Hinton, Jr., R. L. Ziprin, and J. R. DeLoach, 1990b. Effect of dietary lactose on Salmonella colonization of market-age broiler chickens. Avian Dis. 34:668-676. Cox, N. A., J. S. Bailey, J. M. Mauldin, and L. C. Blankenship, 1990a. Presence and impact of Salmonella contamination in commercial broiler hatcheries. Poultry Sci. 69:1606-1609. Cox, N. A., J. S. Bailey, J. M. Mauldin, L. C. Blankenship, and J. L. Wilson, 1991. Extent of salmonellae contamination in breeder hatcheries. Poultry Sci. 70: 416-418.
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ACKNOWLEDGMENTS
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