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Prevention of Salmonella typhimurium Colonization of Broilers with D-Mannose1
Prevention of Salmonella typhimurium Colonization of Broilers with D-Mannose 1 B. A. OYOFO, J. R. DeLOACH,2 D. E. CORRIER, J. O. NORMAN, R. L. ZIPRIN, and H. H. MOLLENHAUER U.S. Department of Agriculture, Agricultural Research Service, Veterinary Toxicology and Entomology Research Laboratory, Rt. 5, Box 810, College Station, Texas 77840 (Received for publication December 2, 1988)
1989 Poultry Science 68:1357-1360
INTRODUCTION
Salmonella typhimurium may cause paratyphoid disease in poultry (Williams, 1972) and is sometimes an enteric pathogen of humans (Center for Disease Control, 1982). In a 5-yr review (1963 to 1967) of human salmonellosis, a close correlation between human salmonellosis and isolates from nonhuman sources was documented (Aserkoff et al, 1970). A 10-yr survey (1970 to 1979) of 475 human salmonellosis outbreaks revealed that poultry food products were the source of 16% of the total outbreaks (Feldman and Riley, 1985). In humans, 5. typhimurium accounted for 35% of the Salmonella isolates in 1984 (Cohen and Tauxe, 1986). Thus, salmonellosis is a human health concern; prevention of S. typhimurium colonization of chickens is important to poultry as well as public health.
'Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable. ^To whom correspondence should be addressed.
The authors' research has recently been directed at preventing Salmonella from colonizing the intestines of broiler chickens. This approach is based on the fact that adherence to the intestinal mucosa is considered a prerequisite for colonization and invasion of hosts by bacteria (Savage, 1972; Gibbons and Van Houte, 1975; Freter, 1981). Thus, blocking adherence may prevent chickens from becoming colonized by S. typhimurium. Several researchers have focused on the mechanism of adherence of bacteria to epithelial cells (Swanson, 1973; Ofek et al., 1975; Beachey and Ofek, 1976; Jones and Freter, 1976). For example, adherence of Escherichia coli to mucosal epithelial cells is mediated by a mannose-specific substance present on the bacterial surface. Consequently, mannose and mannose-derivatives interfere with E. coli adherence to mammalian epithelial cells (Ofek et al., 1977). Type 1 fimbriae may be important for S. typhimurium adherence to intestinal cells (Duguid, 1959; Duguid et al, 1966). Mannose inhibits the agglutination of guinea pig erythrocytes, which condition is used as a test to detect Type 1 fimbriae (Old, 1972). Therefore, mannose may prevent S. typhimurium adherence to intestinal cells. An
1357
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 14, 2015
ABSTRACT Broiler chickens can be contaminated by Salmonella typhimurium, which is a food safety concern. It has been previously shown that D-mannose blocks S. typhimurium adherence to chicken intestine in vitro. One-day-old broiler chickens were fed normal drinking water or drinking water supplemented with 2.5% mannose for 10 days. On Day 3, both groups were challenged orally with 1 x 108 S. typhimurium [ST-10 (Animal Diagnostics Laboratory, Ames, IA)] resistant to Nal and Nov (Sigma, St. Louis, MO). On Day 10 the birds' caecal contents were examined for the antibiotic-marked S. typhimurium. Two additional groups of birds were provided normal drinking water or mannose but were not challenged with the bacteria. Salmonella-challenged control chickens were 78, 82, and 93% colonized whereas Salmonella-challenged mannose-treated chickens were only 28, 21, and 43% colonized. Moreover, the mean logjo counts of control and mannose groups were significantly (P<.001) reduced by at least 99%. Mannose-supplemented drinking water had no effect on weight gains. Certain carbohydrates may provide a means to reduce S. typhimurium contamination in broilers. (Key words: food safety. Salmonella, mannose, colonization, receptors)
1358
OYOFO ET AL. TABLE 1. Experimental design
Group
Treatment1
Day of Salmonella typhimwium challenge
1 2 3 4
Control (water) Control (mannose) Salmonella (water) Salmonella (mannose)
None None 3 3
Days of mannose treatment None 1 to 10 None 1 to 10
n = 30/treatmcnt.
MATERIALS AND METHODS
Chickens. A total of 120 Hubbard x Hubbard male broiler chickens were used per replicate experiment. Birds were obtained from a commercial hatchery and randomly assigned to eight brooder pens (Petersime, Gettysburg, OH) at 15 birds per pen until 10 days of age. Chickens were maintained under fluorescent lighting and given a commercial unmedicated corn-soybean meal-based diet (3,175 kcal ME/ kg; 21.5% CP) ad libitum. The diet was calculated to contain or exceed levels of critical nutrients recommended by the National Research Council (1984). Bacteria. Broth culture of S. typhimurium (ST-10; Animal Diagnostics Laboratory, Ames, IA) was made in 10 mL of trypticase soy broth (Difco, Detroit, MI) grown for 18 to 24 h at 37 C. The strain was screened for mannose sensitivity by the yeast slide agglutination test (Eschdat et al., 1978). This strain (ST-10) was resistant to 20 ng/mL sodium naladixate (Nal) and 25 u,g/mL Novobiocin (Nov, Sigma, St. Louis, MO). Experimental Design. Groups 1 and 3 (30 chickens per group) received tap water and Groups 2 and 4 (30 chickens per group) received 2.5% (wt/vol) mannose in tap water for 10 days (Table 1). On Day 3, Groups 3 and 4 were dosed per os with .25 mL of 5. typhimurium in saline (7.2 x 108 bacteria/mL).
The mannose solution and tap water were renewed daily. Caecal Contents and Weight Gain. Chickens were killed by cervical dislocation on Day 10 or 11 and approximately .2 g of caecal contents was aseptically collected by means of a spatula and diluted 50-fold (wt/vol) in sterile distilled water. Serial (ten-fold) dilutions of the caecal contents were streaked onto brilliant green agar plates containing Nal (20 (Ag/mL) and Nov (25 ng/mL); colonies were counted after 24 to 48 h of incubation at 37 C (Smith and Tucker, 1975). Chickens in all four groups were individually weighed on Days 1 and 10 of the experiment. Statistical Analysis of Data. The experiment was replicated three times. Colonization data were analyzed by chi-square. Classification variables were analyzed with a 2 x 2 contingency table and the chi-square test corrected for continuity (Snedecor and Cochran, 1967; Schlotzhauer and Littell, 1987). The number of Salmonella (Nal, Nov resistant) present in the caecal contents was converted to the log io, as plate count data is not normally distributed. The means, SEM, basic parameter statistics, and the Mann-Whitney U Test were used to determine significant differences between treatment means (Conover, 1971). RESULTS
Effect of D-mannose on colonization of broiler chickens by S. typhimurium was examined. None of the chickens in either of the two non-Salmonella groups were positive for S. typhimurium (Table 2). The Salmonella-challenged (water) group was heavily colonized in each of the three replicates with 78, 82, and 93%, respectively. However, the Salmonella challenged (mannose-treated) groups were significantly (P<.001) less colonized. The mean logio number of viable S. typhimurium per gram of caecal contents was
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 14, 2015
in vitro assay for determining the effect of sugars on adherence of S. typhimurium to chicken intestinal epithelial cells has been developed (Oyofo et al., 1989). In those studies, D-mannose was found to be an effective inhibitor of bacterial adherence. The objective of this study was to determine if 2.5% D-mannose added to the drinking water of broiler chickens would prevent or reduce S. typhimurium colonization in the chicks.
1359
SALMONELLA TYPHIMURIUM COLONIZATION TABLE 2. Effect of D-mannose treatment on Salmonella typhimurium colonization of the caecum of broiler chickens Number of chickens colonized/group1 1st Replication Group
Treatment
n
1 2 3 4
Control (water) Control (mannose) Salmonella (water) Salmonella (mannose)
0 0 22/28 8/29
2nd Replication
% Colonized
n
78 28***
0 0 23/28 6/28
3rd Replication
% Colonized
n
% Colonized
82 21***
0 0 26/28 12/28
93 43***
'The control groups were not analyzed statistically because there were no obvious differences. ***P<.001 between Groups 3 and 4.
DISCUSSION
Provision of D-mannose in drinking water of broiler chickens reduced the percentage of chickens colonized by S. typhimurium (Nal, Nov resistant) from 78% to 28%, 82% to 21%, and 93% to 43%. Moreover, the total number of bacteria per gram of caecal contents was reduced by 99%. These results suggest that the addition of mannose to the drinking water may be a simple means of significantly reducing S. typhimurium colonization in broiler chickens.
Of equal importance is that mannose had no untoward effects on weight gain through the first 10 days of life. By reducing both the bacterial burden and number of birds colonized, the combined effect should translate to cleaner birds at market age. Mannose was used in the present experiment because the in vitro data from previous studies (Oyofo et al., 1989) indicated that mannose was an effective sugar blocker. In vitro adherence is blocked to a lesser extent by other sugars such as galactose (Oyofo, unpublished data). Studies with laboratory animals showed mannose to specifically inhibit the adherence of bacteria including S. typhimurium to intestinal mucosal epithelium (Lindquist et al., 1987). An in vitro assay for detecting 5. typhimurium adherence to chicken intestine was recently used by Oyofo et al. (1989). In those studies, mannose-sensitive strains such as the one used herein adhered more significantly than did mannose-resistant strains. Moreover, adherence could be blocked up to 95% by the addition of 2.5% mannose in the medium. Thus, the in vivo data reported herein are consistent with the in vitro model system.
TABLE 3. Effect of D-mannose on Salmonella typhimurium levels in broilers given oral bacterial dose Number of viable S. typhimurium (Nal, Nov-resistant Strain)1 Group
Treatment
1st Replication
2nd Replication
3rd Replication
3 4
Salmonella (water) Salmonella (mannose)
4.57 ± .21 1.01 ± .60***
4.06 ± .35 -.37 ± .57***
4.86 ± .12 -.82 ± .50***
1 Logio mean number of viable organisms per gram caecal contents ± SEM (N = 28 or 29) for replicate experiments. Nal = sodium naladixate; Nov = Novobiocin; both from Sigma, St. Louis, MO. ***P<.001.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 14, 2015
also determined (Table 3). In three experiments, the log io number of bacteria ranged from 4.07 to 4.86. When the logio number of bacteria in the Salmonella (water) group was compared with the Salmonella (mannose) group, a highly significant (P<.001) difference was found for each replicate experiment. Neither the addition of mannose to drinking water nor the presence of S. typhimurium had a significant effect on weight gains. Group 1, 2, 3, and 4 chickens had weight gains (x ± SD) of 90.3 ± 10.6, 91.1 ± 8.9, 88.8 ± 10.9, and 89.9 ± 10.9 g, respectively.
1360
OYOFO ET AL.
ACKNOWLEDGMENTS
The authors thank Kate Andrews, Clayton Myers, and Bob Droleskey for their technical assistance. REFERENCES Aserkoff, B., S. A. Schroeder, and P. S. Brachman, 1970. Salmonellosis in the United States-A five-year review. Am. J. Epidemiol. 92:13-24. Beachey, E. H., and I. Ofek, 1976. Epithelial cell binding of group A streptococci by lipoteichoic acid fimbriae denuded of M protein. J. Exp. Med. 143:759-771. Center for Disease Control, 1982. Salmonella surveillance. Annual summary, 1980. Center for Disease Control, Atlanta, GA. Cohen, M. L., and R. V. Tauxe, 1986. Drug-resistant Salmonella in the United States: An epidemiologic perspective. Science 234:964-969. Conover, W. J., 1971. Practical Nonparametric Statistics. John Wiley and Sons, Inc., New York, NY. Duguid, J. P., 1959. Fimbriae and adhesive properties in Klebsiella strains. J. Gen. Microbiol. 21:271-286. Duguid, J. P., E. S. Anderson, and I. Campbell, 1966. Fimbriae and adhesive properties in salmonellae. J. Pathol. Bacteriol. 92:107. Duguid, J. P., and R. R. Gillies, 1957. Fimbriae and adhesive properties in dysentery bacilli. J. Pathol. Bacteriol. 74: 397-411. Eschdat, Y., I. Ofek, Y. Yashouv-Gan, N. Sharon, and D. Mirelman, 1978. Isolation of mannose specific lectin from Escherichia coli and its role in the adherence of bacteria to epithelial cells. Biochem. Biophys. Res.
Commun. 85:1551-1559. Feldman, R. A., and L. W. Riley, 1985. Epidemiology of Salmonella and Shigella infections in the United States. Pages 103-116 in: Bacterial Diarrheal Diseases. Y. Takeda and T. Miwatani, ed. KTK Scientific Publishers, Tokyo, Japan. Freter, R., 1981. Mechanism of association of bacteria with mucosal surfaces. CIBA Found. Symp. 80:36-55. Gibbons, R. J., and J. Van Houte, 1975. Bacterial adherence in oral microbial ecology. Ann. Rev. Microbiol. 29: 19-44. Jones, G. W., and R. Freter, 1976. Adhesive properties of Vibrio cholerae: Nature of the interaction with isolated rabbit brush-border membranes and human erythrocytes. Infect. Immun. 14:240-245. Lindquist, B. L., E. Lebenthal, P. C. Lee, M. W. Stinson, and J. M. Merrick, 1987. Adherence of Salmonella typhimurium to small-intestinal enterocytes of the rat. Infect. Immun. 55:3044-3050. National Research Council, 1984. Nutrient Requirements of Poultry. Natl. Academy Sci., Washington, DC. Ofek, I., E. H. Beachey, W. Jefferson, and G. L. Campbell, 1975. Cell membrane-binding properties of group A streptococcal lipoteichoic acid. J. Exp. Med. 141: 990-1003. Ofek, I., D. Mirelman, and N. Sharon, 1977. Adherence of E. coli to human mucosal cells mediated by mannose receptors. Nature 265:623-625. Old, D. C., 1972. Inhibition of the interaction between fimbrial haemagglutinins and erythrocytes by D-mannose and other carbohydrates. J. Gen. Microbiol. 71: 149-157. Oyofo, B. A., R. E. Droleskey, J. O. Norman, H. H. Mollenhauer, R. L. Ziprin, D. E. Corrier, and J. R. DeLoach, 1989. Inhibition by mannose of in vitro colonization of chicken small intestine by Salmonella typhimwium. Poultry Sci. 68:1351-1356. Savage, D. C, 1972. Survival on mucosal epithelia, epithelial penetration and growth in tissues of pathogenic bacteria. Page 25 in: 22nd Symp. Soc. Gen. Microbiol., Cambridge, MA. Schlotzhauer, S. D., and R. C. Littell, 1967. SAS system for elementary statistical analysis. SAS Inst. Inc., Cary, NC. Smith, H. W., and J. F. Tucker, 1975. The effect of antibiotic therapy on the faecal excretion of Salmonella typhimwium by experimentally infected chickens. J. Hyg. 75:275-292. Snedecor, G. W., and W. G. Cochran, 1967. Statistical Methods. 6th ed. Iowa State Univ. Press, Ames, IA. Swanson, J., 1973. Studies on gonococcus infection pili: Their role in attachment of gonococci to tissue culture cells. J. Exp. Med. 137:571-589. Williams, J. E., 1972. Paratyphoid Infections. Page 132 in: Diseases of Poultry. 6th ed. M. S. Hofstad, B. W. Calnek, C. F. Helmboldt, W. M. Reid, and H. W. Yoder, ed. Iowa State Univ. Press, Ames, IA.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 14, 2015
The blocking action of D-mannose of E. coli adherence to epithelial cells is reversible (Duguid and Gillies, 1957; Ofek et al., 1977). Thus, mannose was provided constantly in the drinking water. Previous studies with the in vitro adherence assay suggests that mannose acts at the site of the epithelial cells (Oyofo et al, 1989). The current in vivo studies do not answer the question of whether mannose acts on the epithelial cell or on the bacteria to reduce colonization. From the results of these studies, sugar blockers may offer a valid approach to reducing the percentage of broilers that are colonized by Salmonella and may significantly reduce the public health problem caused by contaminated carcasses at slaughter.
1989 Poultry Science 68:1357-1360
INTRODUCTION
Salmonella typhimurium may cause paratyphoid disease in poultry (Williams, 1972) and is sometimes an enteric pathogen of humans (Center for Disease Control, 1982). In a 5-yr review (1963 to 1967) of human salmonellosis, a close correlation between human salmonellosis and isolates from nonhuman sources was documented (Aserkoff et al, 1970). A 10-yr survey (1970 to 1979) of 475 human salmonellosis outbreaks revealed that poultry food products were the source of 16% of the total outbreaks (Feldman and Riley, 1985). In humans, 5. typhimurium accounted for 35% of the Salmonella isolates in 1984 (Cohen and Tauxe, 1986). Thus, salmonellosis is a human health concern; prevention of S. typhimurium colonization of chickens is important to poultry as well as public health.
'Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable. ^To whom correspondence should be addressed.
The authors' research has recently been directed at preventing Salmonella from colonizing the intestines of broiler chickens. This approach is based on the fact that adherence to the intestinal mucosa is considered a prerequisite for colonization and invasion of hosts by bacteria (Savage, 1972; Gibbons and Van Houte, 1975; Freter, 1981). Thus, blocking adherence may prevent chickens from becoming colonized by S. typhimurium. Several researchers have focused on the mechanism of adherence of bacteria to epithelial cells (Swanson, 1973; Ofek et al., 1975; Beachey and Ofek, 1976; Jones and Freter, 1976). For example, adherence of Escherichia coli to mucosal epithelial cells is mediated by a mannose-specific substance present on the bacterial surface. Consequently, mannose and mannose-derivatives interfere with E. coli adherence to mammalian epithelial cells (Ofek et al., 1977). Type 1 fimbriae may be important for S. typhimurium adherence to intestinal cells (Duguid, 1959; Duguid et al, 1966). Mannose inhibits the agglutination of guinea pig erythrocytes, which condition is used as a test to detect Type 1 fimbriae (Old, 1972). Therefore, mannose may prevent S. typhimurium adherence to intestinal cells. An
1357
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 14, 2015
ABSTRACT Broiler chickens can be contaminated by Salmonella typhimurium, which is a food safety concern. It has been previously shown that D-mannose blocks S. typhimurium adherence to chicken intestine in vitro. One-day-old broiler chickens were fed normal drinking water or drinking water supplemented with 2.5% mannose for 10 days. On Day 3, both groups were challenged orally with 1 x 108 S. typhimurium [ST-10 (Animal Diagnostics Laboratory, Ames, IA)] resistant to Nal and Nov (Sigma, St. Louis, MO). On Day 10 the birds' caecal contents were examined for the antibiotic-marked S. typhimurium. Two additional groups of birds were provided normal drinking water or mannose but were not challenged with the bacteria. Salmonella-challenged control chickens were 78, 82, and 93% colonized whereas Salmonella-challenged mannose-treated chickens were only 28, 21, and 43% colonized. Moreover, the mean logjo counts of control and mannose groups were significantly (P<.001) reduced by at least 99%. Mannose-supplemented drinking water had no effect on weight gains. Certain carbohydrates may provide a means to reduce S. typhimurium contamination in broilers. (Key words: food safety. Salmonella, mannose, colonization, receptors)
1358
OYOFO ET AL. TABLE 1. Experimental design
Group
Treatment1
Day of Salmonella typhimwium challenge
1 2 3 4
Control (water) Control (mannose) Salmonella (water) Salmonella (mannose)
None None 3 3
Days of mannose treatment None 1 to 10 None 1 to 10
n = 30/treatmcnt.
MATERIALS AND METHODS
Chickens. A total of 120 Hubbard x Hubbard male broiler chickens were used per replicate experiment. Birds were obtained from a commercial hatchery and randomly assigned to eight brooder pens (Petersime, Gettysburg, OH) at 15 birds per pen until 10 days of age. Chickens were maintained under fluorescent lighting and given a commercial unmedicated corn-soybean meal-based diet (3,175 kcal ME/ kg; 21.5% CP) ad libitum. The diet was calculated to contain or exceed levels of critical nutrients recommended by the National Research Council (1984). Bacteria. Broth culture of S. typhimurium (ST-10; Animal Diagnostics Laboratory, Ames, IA) was made in 10 mL of trypticase soy broth (Difco, Detroit, MI) grown for 18 to 24 h at 37 C. The strain was screened for mannose sensitivity by the yeast slide agglutination test (Eschdat et al., 1978). This strain (ST-10) was resistant to 20 ng/mL sodium naladixate (Nal) and 25 u,g/mL Novobiocin (Nov, Sigma, St. Louis, MO). Experimental Design. Groups 1 and 3 (30 chickens per group) received tap water and Groups 2 and 4 (30 chickens per group) received 2.5% (wt/vol) mannose in tap water for 10 days (Table 1). On Day 3, Groups 3 and 4 were dosed per os with .25 mL of 5. typhimurium in saline (7.2 x 108 bacteria/mL).
The mannose solution and tap water were renewed daily. Caecal Contents and Weight Gain. Chickens were killed by cervical dislocation on Day 10 or 11 and approximately .2 g of caecal contents was aseptically collected by means of a spatula and diluted 50-fold (wt/vol) in sterile distilled water. Serial (ten-fold) dilutions of the caecal contents were streaked onto brilliant green agar plates containing Nal (20 (Ag/mL) and Nov (25 ng/mL); colonies were counted after 24 to 48 h of incubation at 37 C (Smith and Tucker, 1975). Chickens in all four groups were individually weighed on Days 1 and 10 of the experiment. Statistical Analysis of Data. The experiment was replicated three times. Colonization data were analyzed by chi-square. Classification variables were analyzed with a 2 x 2 contingency table and the chi-square test corrected for continuity (Snedecor and Cochran, 1967; Schlotzhauer and Littell, 1987). The number of Salmonella (Nal, Nov resistant) present in the caecal contents was converted to the log io, as plate count data is not normally distributed. The means, SEM, basic parameter statistics, and the Mann-Whitney U Test were used to determine significant differences between treatment means (Conover, 1971). RESULTS
Effect of D-mannose on colonization of broiler chickens by S. typhimurium was examined. None of the chickens in either of the two non-Salmonella groups were positive for S. typhimurium (Table 2). The Salmonella-challenged (water) group was heavily colonized in each of the three replicates with 78, 82, and 93%, respectively. However, the Salmonella challenged (mannose-treated) groups were significantly (P<.001) less colonized. The mean logio number of viable S. typhimurium per gram of caecal contents was
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 14, 2015
in vitro assay for determining the effect of sugars on adherence of S. typhimurium to chicken intestinal epithelial cells has been developed (Oyofo et al., 1989). In those studies, D-mannose was found to be an effective inhibitor of bacterial adherence. The objective of this study was to determine if 2.5% D-mannose added to the drinking water of broiler chickens would prevent or reduce S. typhimurium colonization in the chicks.
1359
SALMONELLA TYPHIMURIUM COLONIZATION TABLE 2. Effect of D-mannose treatment on Salmonella typhimurium colonization of the caecum of broiler chickens Number of chickens colonized/group1 1st Replication Group
Treatment
n
1 2 3 4
Control (water) Control (mannose) Salmonella (water) Salmonella (mannose)
0 0 22/28 8/29
2nd Replication
% Colonized
n
78 28***
0 0 23/28 6/28
3rd Replication
% Colonized
n
% Colonized
82 21***
0 0 26/28 12/28
93 43***
'The control groups were not analyzed statistically because there were no obvious differences. ***P<.001 between Groups 3 and 4.
DISCUSSION
Provision of D-mannose in drinking water of broiler chickens reduced the percentage of chickens colonized by S. typhimurium (Nal, Nov resistant) from 78% to 28%, 82% to 21%, and 93% to 43%. Moreover, the total number of bacteria per gram of caecal contents was reduced by 99%. These results suggest that the addition of mannose to the drinking water may be a simple means of significantly reducing S. typhimurium colonization in broiler chickens.
Of equal importance is that mannose had no untoward effects on weight gain through the first 10 days of life. By reducing both the bacterial burden and number of birds colonized, the combined effect should translate to cleaner birds at market age. Mannose was used in the present experiment because the in vitro data from previous studies (Oyofo et al., 1989) indicated that mannose was an effective sugar blocker. In vitro adherence is blocked to a lesser extent by other sugars such as galactose (Oyofo, unpublished data). Studies with laboratory animals showed mannose to specifically inhibit the adherence of bacteria including S. typhimurium to intestinal mucosal epithelium (Lindquist et al., 1987). An in vitro assay for detecting 5. typhimurium adherence to chicken intestine was recently used by Oyofo et al. (1989). In those studies, mannose-sensitive strains such as the one used herein adhered more significantly than did mannose-resistant strains. Moreover, adherence could be blocked up to 95% by the addition of 2.5% mannose in the medium. Thus, the in vivo data reported herein are consistent with the in vitro model system.
TABLE 3. Effect of D-mannose on Salmonella typhimurium levels in broilers given oral bacterial dose Number of viable S. typhimurium (Nal, Nov-resistant Strain)1 Group
Treatment
1st Replication
2nd Replication
3rd Replication
3 4
Salmonella (water) Salmonella (mannose)
4.57 ± .21 1.01 ± .60***
4.06 ± .35 -.37 ± .57***
4.86 ± .12 -.82 ± .50***
1 Logio mean number of viable organisms per gram caecal contents ± SEM (N = 28 or 29) for replicate experiments. Nal = sodium naladixate; Nov = Novobiocin; both from Sigma, St. Louis, MO. ***P<.001.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 14, 2015
also determined (Table 3). In three experiments, the log io number of bacteria ranged from 4.07 to 4.86. When the logio number of bacteria in the Salmonella (water) group was compared with the Salmonella (mannose) group, a highly significant (P<.001) difference was found for each replicate experiment. Neither the addition of mannose to drinking water nor the presence of S. typhimurium had a significant effect on weight gains. Group 1, 2, 3, and 4 chickens had weight gains (x ± SD) of 90.3 ± 10.6, 91.1 ± 8.9, 88.8 ± 10.9, and 89.9 ± 10.9 g, respectively.
1360
OYOFO ET AL.
ACKNOWLEDGMENTS
The authors thank Kate Andrews, Clayton Myers, and Bob Droleskey for their technical assistance. REFERENCES Aserkoff, B., S. A. Schroeder, and P. S. Brachman, 1970. Salmonellosis in the United States-A five-year review. Am. J. Epidemiol. 92:13-24. Beachey, E. H., and I. Ofek, 1976. Epithelial cell binding of group A streptococci by lipoteichoic acid fimbriae denuded of M protein. J. Exp. Med. 143:759-771. Center for Disease Control, 1982. Salmonella surveillance. Annual summary, 1980. Center for Disease Control, Atlanta, GA. Cohen, M. L., and R. V. Tauxe, 1986. Drug-resistant Salmonella in the United States: An epidemiologic perspective. Science 234:964-969. Conover, W. J., 1971. Practical Nonparametric Statistics. John Wiley and Sons, Inc., New York, NY. Duguid, J. P., 1959. Fimbriae and adhesive properties in Klebsiella strains. J. Gen. Microbiol. 21:271-286. Duguid, J. P., E. S. Anderson, and I. Campbell, 1966. Fimbriae and adhesive properties in salmonellae. J. Pathol. Bacteriol. 92:107. Duguid, J. P., and R. R. Gillies, 1957. Fimbriae and adhesive properties in dysentery bacilli. J. Pathol. Bacteriol. 74: 397-411. Eschdat, Y., I. Ofek, Y. Yashouv-Gan, N. Sharon, and D. Mirelman, 1978. Isolation of mannose specific lectin from Escherichia coli and its role in the adherence of bacteria to epithelial cells. Biochem. Biophys. Res.
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The blocking action of D-mannose of E. coli adherence to epithelial cells is reversible (Duguid and Gillies, 1957; Ofek et al., 1977). Thus, mannose was provided constantly in the drinking water. Previous studies with the in vitro adherence assay suggests that mannose acts at the site of the epithelial cells (Oyofo et al, 1989). The current in vivo studies do not answer the question of whether mannose acts on the epithelial cell or on the bacteria to reduce colonization. From the results of these studies, sugar blockers may offer a valid approach to reducing the percentage of broilers that are colonized by Salmonella and may significantly reduce the public health problem caused by contaminated carcasses at slaughter.