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Effect of Simultaneous or Delayed Competitive Exclusion Treatment on the Spread of Salmonella in Chicks
01998Applied Poultry Science. Inc
EFFECT OF SIMULTANEOUS OR DELAYED COMPETITIVE EXCLUSION TREATMENT ON THE SPREAD OF SALMONELLA IN CHICKS D. E. CORRIER', J. A. BYRD 11, M. E. HUME, D. J. NISBET, and L. H. STANKER USDA-ARS, Food Animal Protection Research Laboratoy, 2881 F&B Road, College Station, 7X 77845 Phone: (409) 260-9484 F a : (409) 260-9332
Primary Audience: Production Managers, Quality Control Personnel, Researchers
salmonellae may result in colonized seeder DESCRIPTION OF PROBLEM chicks that contaminate feed, water, and litter Reduction of salmonellae contamination of processed poultry products requires an integrated control program implemented at all critical control points from the hatchery through processing [l, 2, 3, 41. Commercial breeder flocks and broiler hatcheries have been identified as sources of salmonellae[5,6]. Exposure of highly susceptible hatchlings to 1 To whom correspondence should be
,
addressed
in the rearing house, resulting in transmission to and colonization of contact birds in the flock during growout [7,8,9].Estimates of the number of seeder chicks that leave the hatchery intestinally colonized with salmonellae range from 5% [9,10] to 9% [ll].Because the colonization dosage of Salmonella cells for day-of-hatch chicks is 100 times less than for
Research Report 133
CORRIER et al. 3-day-old chicks [12], the placement of even 5% seeder chicks in the rearing house may result in the colonization of numerous contact birds during the first days of growout. Recent studies in our laboratory demonstrated that placement of five seeder chicks in floor pens with 95 contacts on the day of hatch resulted in horizontal transmission and cecal colonization of 50% of the contact chicks by Day 17 of growout [B]. Protecting highly susceptible contact chicks against salmonellae exposure from hatchery seeders should contribute to a successful control program. Prophylactic treatment of newly hatched chickswith competitive exclusion cultures (CE) increases resistance to salmonellae colonization [l,2,3,14,15] and may be the best control method currently available during broiler growout [16]. However, CE cultures are most effective when chicks are treated 24 to 48 hr before salmonellae exposure; this treatment provides minimal therapeutic effect in displacing salmonellae from seeder chicks already colonized [17, 18, 19, 201. Although full protection of chicks by CE treatment is not immediate, it has been proposed that treatment may still be advantageous in reducing the horizontal spread of Salmonella to contact chicks exposed simultaneously to colonized seeders (141. The objectives of the present study were to evaluate the protective effect of treatment with a characterized CE culture on horizontal transmission and Salmonella cecal colonization in broiler chicks when Salmonella challenge and CE treatment occur simultaneously, or when CE treatment is delayed for 24 hr after Salmonella challenge.
Division, Rochester, NY) at 625 nm and the number of cells for each inoculum was determined using a standard curve. The viable cell concentration of the challenge inoculum was confirmed by colony counts on brilliant green agar plates (BGA; Oxoid, Unipath Ltd., Basingstoke, Hampshire, England). Media used to culture the NONA-resistant isolate from chicks in experimental studies contained 25pg NO and 20 pug NA/mL to inhibit the growth of other bacteria and nonresistant salmonellae. CHARACTERIZED C O M P E T I T I V E EXCLUSION CULTURE Development of the characterized CE culture using continuous-flow culture systems was described previously [15]. The culture was characterized to contain 29 bacterial isolates composed of 15 facultative anaerobic strains and 14 obligate anaerobic strains representing 10 different genera. The culture, designated continuous-flow-culture 3 (CF3), was prepared and administered to chicks by spray-cabinet application according to the manufacturer’s instructions (Bioscience Division, Milk Specialties Co., Dundee, IL). One hundred chicks were placed in a plastic hatchery tray and sprayed with 20 mL of CF3 culture so as to deliver approximately0.20 mL per chick. The 0.20 mL dosage was previously reported to contain approximately lo7 anaerobic CFU [21]. EXPERIMENT 1 Experiment 1 was conducted to evaluate the protective effect of CF3 culture after Salmonella challenge and simultaneous CF3 treatment. Broiler chicks were obtained on the day of hatch from a commercial hatchery, randomly distributed into four groups of 100 chicks each, and placed in floor pens on new pine shavings litter at a bird density of 0.4 ft2 per chick. The chicks were provided free access to water in 3.8-L plastic drinker jars and an unmedicated corn-soybean meal-based diet that met National Research Council [22] recommendations. The bedding from the chick transport boxes and three 25-g feed samples were determined to be Sulmonelluculture negative using previously described culture procedures [23]. On the day of hatch (Day l), 25 chicks in two groups and 50 chicks in the other two groups were challenged by
MATERIALS AND METHODS SALMONELLA A primary poultry isolate of S. typhimurium obtained from the National Veterinary Services Laboratory (Ames, IA)was selected for resistance to novobiacin (NO) and nalid h c acid (NA) in the authors’ laboratory and maintained in media containing25pg NO and 20pg NNmL. Challenge inocula were prepared from an overnight soy broth culture serially diluted in sterile phosphate-buffered saline. The optical density of the cell dilution was measured with a spectrophotometer (Spec 20D, Milton Roy, Analytical Products
CONTROL OF SALMONELLA SPREAD
134
crop gavage with 0.5 mL of inoculum containing 2 x 1@ NONA-resistant s. typhimurium and designated seeders. The remaining unchallenged chicks in each group were designated contacts. One group containing 25 seeder chicks and one group containing 50 seeders were not treated (controls). All chicks in the remaining two groups containing either 25 or 50 seeders were treated with CF3 culture 1 hr after Salmonella challenge. At 8 days of age, 20 seeder and 20 contact chicks in each group were killed by cervical dislocation and the ceca removed aseptically and cultured for the presence of NONA-resistants. typhimurium. EXPERIMENT 2 Experiment 2 was conducted to evaluate the protective effect of CF3 culture when treatment was delayed for 24 hr after Salmonella challenge. Commercial broiler chicks were obtained on the day of hatch, randomly distributed into two groups of 100 chicks each, placed in floor pens, and provided feed and water per Experiment 1 protocol. On the day of hatch, 25 chicks in each group were challenged by crop gavage with 1.8 x 1@ S. typhimunum and designated seeders. The remaining 75 unchallenged chicks were designated contacts. The two groups of chicks were provided either no treatment (controls) or treated with CF3 culture 24 hr after Salmonella challenge. At 8 days of age, 20 seeder and 20 contact chicks in each group were killed by cervical dislocation and their ceca cultured for the presence of S. lyphimunum. The experimental design was replicated to provide two control groups and two C M treatment groups of 100 chicks per group. PRESENCEOFSALMONELLA INCECAL CONTENTS The ceca from each chick were removed aseptically and a portion (0.2 g) of the cecal contents was diluted in sterile phosphatebuffered saline at l:lO, 1:100, and 1:1,000 dilutions. Each dilution was spread-plated on BGA plates at final dilutions of l:lO, 1:100, 1:1,OOO, and 1:10,O00. The plates were incubated for 24 hr at 37T, and the number of CFU of S. typhimurium was determined using an automatic colony counter (Biotran 111, New Brunswick Scientific, Edison, NJ). cal Salmonella colonies were confirmed by
wi-
biochemical tests on triple sugar iron agar and lysine iron agar, and were further identified as S. typhimurium serologically using Salmonella 0 Antiserum, G r o u p 13 Factors 1,4,12,15 (Difco Laboratories, Detroit, MI). Salmonella colony counts were expressed as loglo Salmonellalg of cecal contents. Cecal contents that were Salmonella-culture negative at the 1:lO dilution were assigned a loglo Salmonella value of 0. STATISTICALANALYSIS Differences in the number of Salmonellacecal-culture-positive chicks between control and treated groups were determined by chisquare analysis [24]. Differences between means were determined using Student's t-test 1251.
RESULTS AND DISCUSSION During Experiment 1, crop gavage of dayold control chicks with lo2 Salmonella resulted in the establishment of colonized seeders in 95-100% of the chicks that were challenged (Table 1). The mean population of Salmonella in the cecal contents of the control seeders at 8 days of age ranged from 5.57 to 6.48 loglo units. The percentage of chicks colonized and the population of Salmonella in the cecal contents of the seeder chicks were similar to those reported previously [7]. Clearly, the increase in Salmonella numbers from the 1@ cells present in the challenge dosage to the high populations present in the ceca at 8 days of age indicated active Salmonella replication in the ceca, resulting in amplification of the low challenge dosage and a ready source of Salmonella exposure for contact chicks in the same pens. Simultaneous CF3 treatment of the challenged chicks on the day of hatch significantly (P5.005) decreased the number of chicks that became Salmonella colonized seeders (Table 1). Although simultaneous treatment was not fully protective, the amplification of the 102 Salmonella challenge dosage was reduced in the treated seeder chicks compared to the controls as indicated by the significantly (Pc .005) lower population of Salmonella in the cecal contents of the treated chicks at 8 days of age (Table 1). The results were similar for both CF3 treated groups of seeder chicks in which either 25 or 50 chicks were challenged per group.
Research Report 135
CORRIER et al.
TABLE 1. Effect of Sdmone Ila m ~ y-t challenge and simultaneous treatment with characterized competitive exclusion culture (CF3) on Salmonella cecal colonization in challenged seeder and unchallenged contact chicksA
I Control
SEEDER CHICKS
SEEDER PER PEN
CONTACT CHICKS
Salmonella Positive Loglo Salmonella/g Salmonella Positive Loglo Salmonella/g 25
ChicksDotal(%)
Cecal Contents
Chicks/Total (%)
Cecal Contents
20/20 (loo)
6.48f 0.80'
14/20 (70)
2.86k2.51B
CF3 Treated
25
7/14 (50).
1.73k1.94'
1/20 (5)'
0.32 k 1.45.
Control
50
19/20 (95)
5.57k1.91
16/20 (80)
3.34 22.02
CF3 Treated
50
12/20 (60).
2.1222.03.
oi20 (0)
OfO'
Previous studies have demonstrated that CE treatment of contact chicks on the day of hatch followed by the establishment of a seeder bird challenge on Day 2 [26] or on Day 3 [27] significantly protects the contact chicks against Salmonella transmission. It has also been reported that challenge of newly hatched chicks with 104 S. enterifidis (PT4) and simultaneous CE treatment failed to protect the challenged chicks against cecal colonization but did reduce the horizontal spread ofSalmonella to treated contacts and the population of Salmonella in contact chick ceca [14].The seeder:contact ratio in that study was reported at 1:15.During Experiment 1of the present study, the contact chickswere exposed to a seedercontact ratio of 1:3 or 1:l and treated simultaneously with CF3 culture on the day of hatch. The number of Salmonellacecal-culture-positive chicks decreased significantly (Ps.005)from 70430% in the untreated contacts to 0 or 5% in the treated
contacts (Table 1).The population of Salmonella in the ceca of the treated contact chicks was also reduced significantly (PS ,005)compared to that of the untreated contact chicks. The results indicated that at seeder rates of either 25 or 50%, simultaneousCF3 treatment of contact chicks markedly reduced the horizontal spread of Salmonella from seeder to contact pen mates. During Experiment 2, the incidence of Salmonella cecal colonization and the population of Salmonella in the ceca did not differ significantly between either the replicate control groups or the replicate treatment groups. Data from the two replicates were, therefore, combined and analyzed statistically (Table 2). Much as in Experiment 1, challenge of day-old control chicks with lo2 Salmonella resulted in the establishment of colonized seeders in 100% of the chicks that were challenged. The high mean population of Salmonella in the cecal contents of these control seeders
TABLE 2. Effect of Salmonelk m h i m u r i m challenge and l d a y delayed treatment with characterized competitive exclusion culture (CF3) on m o n e lla cecal colonization in challenged seeder and unchallenged contact chicksA
I
GROUP
SEEDER CHICKS
CONTACT CHICKS I
SalmonellaPositive ChicksKotal (%)
L.ogloSalmonella/g Cecal Contents
Control
40/40 (100)
6.41rt1.0SB
38/40 (95)
5.442 l.MB
CF3 Treated
37/40 (93)
4.41f1.81'
24/40 (60) *
2.69k2.49'
Positive Chicksmotal (YO)
Loglo Salmonella/g Cecal Contents
I
JAPR CONTROL OF SALMONELLA SPREAD
136
(6.41 loglo units) at 8 days of age indicated Salmonella replication in the ceca and amplification of the low 102 Salmonella challenge dosage (Table 2). CF3 treatment 24 hr after challenge failed to significantly affect the incidence of cecal colonization and Salmonella seeder establishment (Table 2). Delayed treatment did, however, significantly (PS .005)decrease the Salmonella population in the ceca of the challenged seeder chicks, although the number of Salmonella still present at 8 days of age remained high. The results support previous studies that have demonstrated that early exposure to Salmonella in the hatchery limits the effectiveness of CE treatment [7,17, 18, 19, 201. Although limited in effectiveness,
delayed CF3 treatment did measurably decrease the spread of Salmonella from the seeder chicks to contact chicks and reduced the population ofSalmonella in the ceca of the contacts (Table 2). A comparison of the results from Experiment 1 and Experiment 2 indicates that CF3 treatment was more effective when administered simultaneously with Salmonella challenge than after a 24-hr delay. When CF3 treatment occurred simultaneously with Salmonella challenge, seeder establishment was decreased by 35-50% and the spread of Salmonella from seeder chicks to highly susceptible contact chicks was markedly reduced.
CONCLUSIONS AND APPLICATIONS 1. Hatchling chicks in contact with Salmonella seeder chicks are highly susceptible to Salmonella transmission. 2. Treatment of chicks with a competitive exclusion culture simultaneously with Salmonella challenge reduces the establishment of seeder chicks and effectively controls Salmonella transmission to contact chicks. 3. Treatment with a competitive exclusion culture 24 hr after Salmonella exposure fails to prevent the establishment of seeder chicks but can help to reduce Salmonella spread to contact chicks.
REFERENCES AND NOTES 1. Stavric, S. and J.Y. D’aoust, 1993. Undefined and defined bacterial preparations for the competitive exclusion of Salmonella in poultry - A review. J. Food Prot. 56:173-180. 2. Goren, E, W.A. de Jon& P. Doornenbal, N.M. Bolder, R.W.A.W. Mulder, and A. Jansen, 1988. Reduction of Salmonellainfection of broilers by spray application of intestinal microflora: A longitudinal study. Vet. Q. 10249-255. 3. Blankenship, LC., J.S. Bailey, N.A. Cox, N.J.Stern, R. Brewer, and 0. Williams, 1993. Two-step mucosal competitive exclusion flora treatment to diminish salmonellae in commercial broiler chickens. Poultry Sci. 72~1667-1672.
4. Rigby, C.E and J.R Pettit, 1980. Changes in the Salmonella status of broiler chickens subjected to simulated shipping conditions. Can. J. Comp. Med. 44:374381.
8. Cason, J.k,N.A. Cox, and J.S. Bailey, 1994.Transmission of Salmonella tMhlmunum during hatching of broiler chicks. Avian Dis. 38583-588. 9. Bailey, J.S., N.A. Cox, and M.E Berrang, 1994. Hatchery-acquired salmonellae in broiler chicks. Poultry Sci. 73:1153-1157. 10. Lahellec, C. and P. Colin, 1985. Relationship between serotypes ofsalmonellae from hatcheries and rearing farms and those from processed poultry carcasses. Br. Poultry Sci. 26:179-186. 11. Jones, F.C., R.C. Axtell, F.R Tamer, D.V. Rives, S.E Scheideler, and M.J. Wineland, 1991. Environmental factors contributing to salmonellae colonizatjon of chickens. Pages 3-20 in: Colonization Control of Human Bacterial Enteropathogens in Poultry. L.C. Blankenship, ed. Academic Press, Inc., San Diego. CA.
Blankenship, 1990. Presence and impact of Salmonella contamination in commercial broiler hatcheries. Poultry Sci. 69:1606-1609.
12. Cox, N.A., J.S. Bailey, LC. Blankenship, R.J. Meinersrnan, N.J. Stern, and F. McHan, 1990. Colonization dose 50% values for Salmonellaadministered orally and intracloacally to young broiler chicks. Poultry Sci. 691809-1812.
6. Cox, N.A., J.S. Bailey, J.M. Mauldin, LC. Blankenship, and J.L. Wilson, 1991. Extent ofsalmonella contam. ination of breeder hatcheries. Poultry Sci. 70:416418.
13. Byrd, J.A., Food Animal Protection Research Laboratory, USDA-ARS, 2881 F&B Road, College Station, TX 77845. Personal communication.
7. Cox, N.A., J.S. Bailey, and M.E Berrang, 1996. Alternative routes for Salmonellaintestinal tract colonization of chicks. J. Appl. Poultry Res. 5:282-288.
14. Mead, G.C., 1991. Developments in competitive exclusion to control -caniage in poultry. Pages 91-104 in: Colonization Control of Human Bacterial En-
5. Cox, N.A., J.S. Bailey, J.M. Mauldin, and LC.
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CORRIER el al. teropatho e n s in Poultry. L.C. Blankenship, ed. Academic tress Inc., San Diego, CA. 15. Corrier, D.E, D.J. Nisbet, C.M. Scanlan, A.G. HoUister!and J.R DeLoach, 1995. Control of S.ahu@a colonization in broiler chicks with a contin%%%%aracterized mixed culture of cecal bacteria. Poultry Sci. 74:916-924. 16. Bailey, J.S., 1987. Factors affecting microbial competitive exclusion in poultv. Food Tech. 41:8&92.
21. Corrier, D.E., D.J. Nisbel, N.K. Keith, ED. Dunlap, and J.R DeLoach, 1996. S ray application and dosage titration of characterized C& competitive exclusion culture for w o n e l l a control in broiler chickens. Pages 272-275 in: Proc. 45th Western Poultry Dis. Conf., Cancun, Mexico. 22. National Research Council, 1984. Nutrient Reuirements of Poult . 8th Rev. Edition. Natl. Acad. %ress,Washington, D?.
5,1979. Sensitivity of young chickens to
23. Andmws, W.H., V.R. Bruce, G. June, F. Satchell, and P. Sherrod, 1992. Salmonella. Pages 51-69 in: Bacteriological Analytical Manual. 7th Edition. Assn. Offic. Analy. Chemists, Arlington, VA.
18. Soerjadi, AS., S.M. Stehman, G.H. Snoeyenbos, O.M. Weinack, and C.F. Smyser, 1981. Some measurements of protection against paratyphoid and ' ' b competitive exclusion in chickens. -25:707-!12.
24. Luginbuke, R.C. and S.D. Schlotzhauer, 1987. SAWSTAT Guide for Personal Computers. 6th Edition. SAS Institute, Inc., Cary, NC.
17. Seuna,
SalnlQnella
var. copenhagen and S. infant& infection a n e t i v e effect of cultured intestinal microflora. Avian Dis. 23392-400.
19. Hudault, S., H. Bewa, C. Bridonneau, and P. Raibaud, 1985. Efficacy of various bacterial suspensions derived from cecal floras of conventional chickens in reducing the population level of in gnotobiotic mice and chicken intesti%?%% Microbiol. 31:832-838.
m,
20. Impey, C.S., G.C. Mead,and M. Hinton, 1987. Influence of continuous challengevia the feed on competitive exclusion of salmonellas from broiler chicks. J. Appl. Bacteriol. 63139-146.
25. Snedecor, G.W. and W.G. Cochran, 1967. Statistical Methods. 6th Edition. Iowa State Univ. Press, Ames, IA. 26. Schneilz, C., 1992. Automated droplet application of a competitive exclusion preparation. Poultry Sci. 71:2125-2128. 27. Coder, D.E, B. Hargis, A. Hinton, Jr., D. Lindsey, D. Caldwell, J. Manning, and J.R DeLoach, 1991. Effect of anaerobic cecal microflora and dietary lactose on colonization resistance of layer chicks to invasive S a l m o n e l l a m . Avian Dis. 35:337-343.
EFFECT OF SIMULTANEOUS OR DELAYED COMPETITIVE EXCLUSION TREATMENT ON THE SPREAD OF SALMONELLA IN CHICKS D. E. CORRIER', J. A. BYRD 11, M. E. HUME, D. J. NISBET, and L. H. STANKER USDA-ARS, Food Animal Protection Research Laboratoy, 2881 F&B Road, College Station, 7X 77845 Phone: (409) 260-9484 F a : (409) 260-9332
Primary Audience: Production Managers, Quality Control Personnel, Researchers
salmonellae may result in colonized seeder DESCRIPTION OF PROBLEM chicks that contaminate feed, water, and litter Reduction of salmonellae contamination of processed poultry products requires an integrated control program implemented at all critical control points from the hatchery through processing [l, 2, 3, 41. Commercial breeder flocks and broiler hatcheries have been identified as sources of salmonellae[5,6]. Exposure of highly susceptible hatchlings to 1 To whom correspondence should be
,
addressed
in the rearing house, resulting in transmission to and colonization of contact birds in the flock during growout [7,8,9].Estimates of the number of seeder chicks that leave the hatchery intestinally colonized with salmonellae range from 5% [9,10] to 9% [ll].Because the colonization dosage of Salmonella cells for day-of-hatch chicks is 100 times less than for
Research Report 133
CORRIER et al. 3-day-old chicks [12], the placement of even 5% seeder chicks in the rearing house may result in the colonization of numerous contact birds during the first days of growout. Recent studies in our laboratory demonstrated that placement of five seeder chicks in floor pens with 95 contacts on the day of hatch resulted in horizontal transmission and cecal colonization of 50% of the contact chicks by Day 17 of growout [B]. Protecting highly susceptible contact chicks against salmonellae exposure from hatchery seeders should contribute to a successful control program. Prophylactic treatment of newly hatched chickswith competitive exclusion cultures (CE) increases resistance to salmonellae colonization [l,2,3,14,15] and may be the best control method currently available during broiler growout [16]. However, CE cultures are most effective when chicks are treated 24 to 48 hr before salmonellae exposure; this treatment provides minimal therapeutic effect in displacing salmonellae from seeder chicks already colonized [17, 18, 19, 201. Although full protection of chicks by CE treatment is not immediate, it has been proposed that treatment may still be advantageous in reducing the horizontal spread of Salmonella to contact chicks exposed simultaneously to colonized seeders (141. The objectives of the present study were to evaluate the protective effect of treatment with a characterized CE culture on horizontal transmission and Salmonella cecal colonization in broiler chicks when Salmonella challenge and CE treatment occur simultaneously, or when CE treatment is delayed for 24 hr after Salmonella challenge.
Division, Rochester, NY) at 625 nm and the number of cells for each inoculum was determined using a standard curve. The viable cell concentration of the challenge inoculum was confirmed by colony counts on brilliant green agar plates (BGA; Oxoid, Unipath Ltd., Basingstoke, Hampshire, England). Media used to culture the NONA-resistant isolate from chicks in experimental studies contained 25pg NO and 20 pug NA/mL to inhibit the growth of other bacteria and nonresistant salmonellae. CHARACTERIZED C O M P E T I T I V E EXCLUSION CULTURE Development of the characterized CE culture using continuous-flow culture systems was described previously [15]. The culture was characterized to contain 29 bacterial isolates composed of 15 facultative anaerobic strains and 14 obligate anaerobic strains representing 10 different genera. The culture, designated continuous-flow-culture 3 (CF3), was prepared and administered to chicks by spray-cabinet application according to the manufacturer’s instructions (Bioscience Division, Milk Specialties Co., Dundee, IL). One hundred chicks were placed in a plastic hatchery tray and sprayed with 20 mL of CF3 culture so as to deliver approximately0.20 mL per chick. The 0.20 mL dosage was previously reported to contain approximately lo7 anaerobic CFU [21]. EXPERIMENT 1 Experiment 1 was conducted to evaluate the protective effect of CF3 culture after Salmonella challenge and simultaneous CF3 treatment. Broiler chicks were obtained on the day of hatch from a commercial hatchery, randomly distributed into four groups of 100 chicks each, and placed in floor pens on new pine shavings litter at a bird density of 0.4 ft2 per chick. The chicks were provided free access to water in 3.8-L plastic drinker jars and an unmedicated corn-soybean meal-based diet that met National Research Council [22] recommendations. The bedding from the chick transport boxes and three 25-g feed samples were determined to be Sulmonelluculture negative using previously described culture procedures [23]. On the day of hatch (Day l), 25 chicks in two groups and 50 chicks in the other two groups were challenged by
MATERIALS AND METHODS SALMONELLA A primary poultry isolate of S. typhimurium obtained from the National Veterinary Services Laboratory (Ames, IA)was selected for resistance to novobiacin (NO) and nalid h c acid (NA) in the authors’ laboratory and maintained in media containing25pg NO and 20pg NNmL. Challenge inocula were prepared from an overnight soy broth culture serially diluted in sterile phosphate-buffered saline. The optical density of the cell dilution was measured with a spectrophotometer (Spec 20D, Milton Roy, Analytical Products
CONTROL OF SALMONELLA SPREAD
134
crop gavage with 0.5 mL of inoculum containing 2 x 1@ NONA-resistant s. typhimurium and designated seeders. The remaining unchallenged chicks in each group were designated contacts. One group containing 25 seeder chicks and one group containing 50 seeders were not treated (controls). All chicks in the remaining two groups containing either 25 or 50 seeders were treated with CF3 culture 1 hr after Salmonella challenge. At 8 days of age, 20 seeder and 20 contact chicks in each group were killed by cervical dislocation and the ceca removed aseptically and cultured for the presence of NONA-resistants. typhimurium. EXPERIMENT 2 Experiment 2 was conducted to evaluate the protective effect of CF3 culture when treatment was delayed for 24 hr after Salmonella challenge. Commercial broiler chicks were obtained on the day of hatch, randomly distributed into two groups of 100 chicks each, placed in floor pens, and provided feed and water per Experiment 1 protocol. On the day of hatch, 25 chicks in each group were challenged by crop gavage with 1.8 x 1@ S. typhimunum and designated seeders. The remaining 75 unchallenged chicks were designated contacts. The two groups of chicks were provided either no treatment (controls) or treated with CF3 culture 24 hr after Salmonella challenge. At 8 days of age, 20 seeder and 20 contact chicks in each group were killed by cervical dislocation and their ceca cultured for the presence of S. lyphimunum. The experimental design was replicated to provide two control groups and two C M treatment groups of 100 chicks per group. PRESENCEOFSALMONELLA INCECAL CONTENTS The ceca from each chick were removed aseptically and a portion (0.2 g) of the cecal contents was diluted in sterile phosphatebuffered saline at l:lO, 1:100, and 1:1,000 dilutions. Each dilution was spread-plated on BGA plates at final dilutions of l:lO, 1:100, 1:1,OOO, and 1:10,O00. The plates were incubated for 24 hr at 37T, and the number of CFU of S. typhimurium was determined using an automatic colony counter (Biotran 111, New Brunswick Scientific, Edison, NJ). cal Salmonella colonies were confirmed by
wi-
biochemical tests on triple sugar iron agar and lysine iron agar, and were further identified as S. typhimurium serologically using Salmonella 0 Antiserum, G r o u p 13 Factors 1,4,12,15 (Difco Laboratories, Detroit, MI). Salmonella colony counts were expressed as loglo Salmonellalg of cecal contents. Cecal contents that were Salmonella-culture negative at the 1:lO dilution were assigned a loglo Salmonella value of 0. STATISTICALANALYSIS Differences in the number of Salmonellacecal-culture-positive chicks between control and treated groups were determined by chisquare analysis [24]. Differences between means were determined using Student's t-test 1251.
RESULTS AND DISCUSSION During Experiment 1, crop gavage of dayold control chicks with lo2 Salmonella resulted in the establishment of colonized seeders in 95-100% of the chicks that were challenged (Table 1). The mean population of Salmonella in the cecal contents of the control seeders at 8 days of age ranged from 5.57 to 6.48 loglo units. The percentage of chicks colonized and the population of Salmonella in the cecal contents of the seeder chicks were similar to those reported previously [7]. Clearly, the increase in Salmonella numbers from the 1@ cells present in the challenge dosage to the high populations present in the ceca at 8 days of age indicated active Salmonella replication in the ceca, resulting in amplification of the low challenge dosage and a ready source of Salmonella exposure for contact chicks in the same pens. Simultaneous CF3 treatment of the challenged chicks on the day of hatch significantly (P5.005) decreased the number of chicks that became Salmonella colonized seeders (Table 1). Although simultaneous treatment was not fully protective, the amplification of the 102 Salmonella challenge dosage was reduced in the treated seeder chicks compared to the controls as indicated by the significantly (Pc .005) lower population of Salmonella in the cecal contents of the treated chicks at 8 days of age (Table 1). The results were similar for both CF3 treated groups of seeder chicks in which either 25 or 50 chicks were challenged per group.
Research Report 135
CORRIER et al.
TABLE 1. Effect of Sdmone Ila m ~ y-t challenge and simultaneous treatment with characterized competitive exclusion culture (CF3) on Salmonella cecal colonization in challenged seeder and unchallenged contact chicksA
I Control
SEEDER CHICKS
SEEDER PER PEN
CONTACT CHICKS
Salmonella Positive Loglo Salmonella/g Salmonella Positive Loglo Salmonella/g 25
ChicksDotal(%)
Cecal Contents
Chicks/Total (%)
Cecal Contents
20/20 (loo)
6.48f 0.80'
14/20 (70)
2.86k2.51B
CF3 Treated
25
7/14 (50).
1.73k1.94'
1/20 (5)'
0.32 k 1.45.
Control
50
19/20 (95)
5.57k1.91
16/20 (80)
3.34 22.02
CF3 Treated
50
12/20 (60).
2.1222.03.
oi20 (0)
OfO'
Previous studies have demonstrated that CE treatment of contact chicks on the day of hatch followed by the establishment of a seeder bird challenge on Day 2 [26] or on Day 3 [27] significantly protects the contact chicks against Salmonella transmission. It has also been reported that challenge of newly hatched chicks with 104 S. enterifidis (PT4) and simultaneous CE treatment failed to protect the challenged chicks against cecal colonization but did reduce the horizontal spread ofSalmonella to treated contacts and the population of Salmonella in contact chick ceca [14].The seeder:contact ratio in that study was reported at 1:15.During Experiment 1of the present study, the contact chickswere exposed to a seedercontact ratio of 1:3 or 1:l and treated simultaneously with CF3 culture on the day of hatch. The number of Salmonellacecal-culture-positive chicks decreased significantly (Ps.005)from 70430% in the untreated contacts to 0 or 5% in the treated
contacts (Table 1).The population of Salmonella in the ceca of the treated contact chicks was also reduced significantly (PS ,005)compared to that of the untreated contact chicks. The results indicated that at seeder rates of either 25 or 50%, simultaneousCF3 treatment of contact chicks markedly reduced the horizontal spread of Salmonella from seeder to contact pen mates. During Experiment 2, the incidence of Salmonella cecal colonization and the population of Salmonella in the ceca did not differ significantly between either the replicate control groups or the replicate treatment groups. Data from the two replicates were, therefore, combined and analyzed statistically (Table 2). Much as in Experiment 1, challenge of day-old control chicks with lo2 Salmonella resulted in the establishment of colonized seeders in 100% of the chicks that were challenged. The high mean population of Salmonella in the cecal contents of these control seeders
TABLE 2. Effect of Salmonelk m h i m u r i m challenge and l d a y delayed treatment with characterized competitive exclusion culture (CF3) on m o n e lla cecal colonization in challenged seeder and unchallenged contact chicksA
I
GROUP
SEEDER CHICKS
CONTACT CHICKS I
SalmonellaPositive ChicksKotal (%)
L.ogloSalmonella/g Cecal Contents
Control
40/40 (100)
6.41rt1.0SB
38/40 (95)
5.442 l.MB
CF3 Treated
37/40 (93)
4.41f1.81'
24/40 (60) *
2.69k2.49'
Positive Chicksmotal (YO)
Loglo Salmonella/g Cecal Contents
I
JAPR CONTROL OF SALMONELLA SPREAD
136
(6.41 loglo units) at 8 days of age indicated Salmonella replication in the ceca and amplification of the low 102 Salmonella challenge dosage (Table 2). CF3 treatment 24 hr after challenge failed to significantly affect the incidence of cecal colonization and Salmonella seeder establishment (Table 2). Delayed treatment did, however, significantly (PS .005)decrease the Salmonella population in the ceca of the challenged seeder chicks, although the number of Salmonella still present at 8 days of age remained high. The results support previous studies that have demonstrated that early exposure to Salmonella in the hatchery limits the effectiveness of CE treatment [7,17, 18, 19, 201. Although limited in effectiveness,
delayed CF3 treatment did measurably decrease the spread of Salmonella from the seeder chicks to contact chicks and reduced the population ofSalmonella in the ceca of the contacts (Table 2). A comparison of the results from Experiment 1 and Experiment 2 indicates that CF3 treatment was more effective when administered simultaneously with Salmonella challenge than after a 24-hr delay. When CF3 treatment occurred simultaneously with Salmonella challenge, seeder establishment was decreased by 35-50% and the spread of Salmonella from seeder chicks to highly susceptible contact chicks was markedly reduced.
CONCLUSIONS AND APPLICATIONS 1. Hatchling chicks in contact with Salmonella seeder chicks are highly susceptible to Salmonella transmission. 2. Treatment of chicks with a competitive exclusion culture simultaneously with Salmonella challenge reduces the establishment of seeder chicks and effectively controls Salmonella transmission to contact chicks. 3. Treatment with a competitive exclusion culture 24 hr after Salmonella exposure fails to prevent the establishment of seeder chicks but can help to reduce Salmonella spread to contact chicks.
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