RESEARCH NOTES Research Note: In Ovo Administration of a Competitive Exclusion Culture Treatment to Broiler Embryos N. A. COX,1 J. S. BAILEY, L. C. BLANKENSHIP, and R. P. GILDERSLEEVE2 USDA, Agricultural Research Service, Russell Research Center, P.O. Box 5677, Athens, Georgia 30613 and
ABSTRACT Exposure of chicks to salmonellae in the hatchery and hatchery environment limits the effectiveness of a competitive exclusion (CE) culture treatment. Therefore, in an attempt to apply treatment before chicks are exposed to salmonellae, the CE culture was introduced in ovo to unhatched embryos. An undefined, anaerobically grown CE culture, derived from cecal contents of healthy adult chickens, was diluted 1:1,000 or 1:1,000,000 and inoculated either into the air cell or beneath the inner air cell membrane of 17-day-old incubating hatching eggs. The treated chicks were more resistant than untreated chicks to varying challenge levels of Salmonella typhimurium, indicating that it may be possible to initiate protection of chicks to salmonellae challenge prior to hatching into a contaminated environment. (Key words: eggs, Salmonella, broiler chicks, colonization resistance, competitive exclusion) 1992 Poultry Science 71:1781-1784
sion (CE) or the Nurmi concept. Since the early 1970s, the efficacy of CE has been Research studies in Finland in the early demonstrated in a number of laboratories 1970s suggested that susceptibility of the (Snoeyenbos et at, 1978; Barnes et al, 1980; newly hatched broiler chick to salmonellae Pivnick et al., 1981; Bailey et al, 1988). colonization was due to delayed establish- Although the efficacy of CE has been ment of normal gut microflora in chicks clearly demonstrated in the laboratory, the reared according to modern mass produc- limited number of large-scale field trials tion methods (Nurmi and Rantala, 1973). that have been conducted have produced They also demonstrated that salmonellae mixed results (Huttner et al, 1981; Weirup infections could be prevented by feeding et al, 1987; Goren et al, 1988). anaerobically grown cultures obtained At hatch, most chicks have very little from the intestinal flora of adult fowl to microflora in their gut and are highly newly hatched chicks (Nurmi and Rantala, susceptible to intestinal colonization by 1973; Rantala and Nurmi, 1973). This salmonellae (Pivnick and Nurmi, 1982). By technique is known as competitive exclu- 2 wk of age, chicks have a mature gut microflora (Barnes et al, 1972) and are therefore much more resistant to intestinal colonization by salmonellae. Competitive Received for publication January 21, 1992. exclusion cultures given to chicks on the Accepted for publication June 19, 1992. day of hatch speed maturation of gut ! To whom correspondence should be addressed. 2 Embrex Inc., P.O. Box 13989, Research Triangle microflora and within a few hours significantly increase the resistance of chicks to Park, NC 27709. INTRODUCTION
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Embrex Inc., P.O. Box 13989, Research Triangle Park, North Carolina 27709
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COX ET AL.
salmonellae (Seuna, 1979; Soerjadi et al, 1981). Commercial hatchery environments have been shown to be significantly contaminated with salmonellae (Cox et al, 1990b, 1991), which can limit the effectiveness of CE treatment (Goren et al, 1988; E. Nurmi, National Veterinary Institute, Helsinki, Finland, personal communication, 1989). The objective of the present study was to assess the benefit of applying CE treatment prior to environmental exposure of chicks to salmonellae.
Competitive Exclusion Culture Fecal and cecal droppings were collected from 1-yr-old, salmonellae-free, caged layer chickens. These droppings (.5 g) were inoculated into 10 mL of Viande-Levure (VL) broth (Bailey et al, 1988) and incubated anaerobically at 35 C for 48 h. This culture (.2 mL) was subcultured into 10 mL of VL broth and similarly incubated anaerobically. Following incubation, glycerol was added to the broth at a final concentration of 15%. This culture with glycerol was divided into 1.0-mL portions, displaced into small plastic vials, and frozen at -70 C. For each experiment, a vial of the frozen culture was thawed and .2 mL of the culture was used to inoculate 10 mL of VL broth, which was then incubated for 48 h at 35 C. Salmonellae Chicks were challenged with a nalidixicacid-resistant strain of Salmonella typhimurium prepared according to the procedures of Bailey et al. (1988). Challenge levels were either 103, 1CP, or 107 per chick. In Ovo Experiments In ovo treatment with CE cultures was made directly onto the inner air cell shell membrane (AC) or beneath the inner membrane (BM) of 18-day-old incubating embryos. Three days post-treatment (Day 21 of incubation), control chicks and chicks treated on the day of hatch in each experiment were orally challenged by gavage with varying numbers of Salmonella
3
Sanofi Animal Health Inc., Berlin, MD 21811.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on January 21, 2015
MATERIALS AND METHODS
typhimurium cells. Seven days postchallenge, chicks were euthanatized by CO2 asphyxiation and then soaked in 70% ethyl alcohol for 1 min to reduce external bacterial contamination. Both ceca from each chick were then excised aseptically and analyzed according to the swab plate method described by Bailey et al. (1988) for recovery of the nalidixic acid marker resistant strain of S. typhimurium. In Ovo Experiment 1. This experiment was conducted as a pilot study. On Day 18 of incubation, 700 commercial broiler eggs containing live embryos, as determined by candling, were removed from an incubator and divided at random into seven groups. Untreated control eggs in the first group were transferred to a hatching basket. Eggs in the second group were injected with undiluted CE culture at the time of transfer from the setter to the hatcher. The shell was pierced at the pole of the air cell end of the egg with an 18-gauge hypodermic needle inserted to a depth of 2 to 5 mm past the shell surface. The 18-gauge needle was withdrawn and a syringe with a 22-gauge hypodermic needle was inserted into the hole in the shell to a depth of 2 to 5 mm. Also, 100 JUL of undiluted CE culture was injected onto the AC membrane. Eggs comprising the third and fourth treatment groups received 100 |iL of 1:1,000 or 1:1,000,000 diluted CE culture, respectively. Sanofi Marek's diluent3 was used to dilute the CE culture. The shells of the eggs in the remaining groups were pierced as described above, but during transfer were injected with a 22-gauge needle to a depth of 20 to 25 mm beneath the shell in order to deliver the CE culture BM. Eggs in the fifth, sixth, and seventh treatment groups were injected with 100 uL of undiluted CE culture, 1:1,000 diluted CE culture, and 1: 1,000,000 diluted CE culture, respectively. Eggs were returned to the incubator for 3 additional days of incubation. Hatchability of each group was noted and expressed as the percentage of live chicks. Hatched chicks in the control and several treatment groups were orally challenged with 106 S. typhimurium cells and evaluated for resistance to colonization as previously described.
RESEARCH NOTE
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a_d
Statistical Analyses Frequency data (including hatchability) from each experiment were subjected to chisquare analysis (Statistical Package for the Social Sciences, 1988). In Experiment 2, frequency data were pooled across replicates and the pooled data were analyzed. A significance level of P < .05 was selected. RESULTS AND DISCUSSION When varying dilutions of anaerobically grown, CE culture was administered in ovo in the AC or BM, only the 1:1,000 and 1:1,000,000 dilutions administered into the air cell resulted in hatchabilities approaching commercial acceptability (Table 1). Only the chicks inoculated in ovo onto the air cell with a 1:1,000 dilution of the CE culture acquired resistance to a 106 cell oral challenge of S. typhimurium on the day of hatch when compared with the untreated controls. Because the number of chicks in the first experiment was limited, a second series of experiments was conducted utilizing a greater number of day-of-hatch chicks with varying Salmonella challenge dosages (Table 2). In Trial 1 of these studies, in ovo introduction of a 1:1,000 dilution of CE culture onto AC of Day 18 embryonated eggs produced day-of-hatch chicks completely resistant to 103 and 105 S. typhimurium challenge. In addition, only four of eight chicks treated with CE culture were colonized when challenged with 107 S. typhimurium, whereas all of the untreated chicks were colonized. The colonization resistance to challenge was less evident in Trial 2 than in Trial 1.
Values in a column with no common superscripts differ significantly (P < .05). !Number of normal chicks hatched out of 100 fertile eggs set. 2 The air cell was the site of injection with undiluted 48-h VL culture. 3 ND = not done. 4 Same as (2) except that the VL culture was diluted 1:1,000. 'Same as (2) except that the VL culture was diluted 1:1,000,000. 6 Injection was beneath the inner air cell membrane of the egg with undiluted culture. 7 Same as (6) except that the VL culture was diluted 1:1,000. 8 Same as (6) except that the VL culture was diluted 1:1,000,000.
Pooled data revealed significantly different resistances among treated chicks to challenges of S. typhimurium after in ovo injection of a 1:1,000 dilution. Competitive exclusion has been demonstrated in laboratories in various countries to be an effective method of reducing salmonellae colonization in the intestinal tract of chickens (Snoeyenbos et ah, 1978; Blanchfield et ah, 1982; Bailey et ah, 1988). The presence and persistence of salmonellae in commercial hatcheries (Cox et ah, 1990, 1991) limits the effectiveness of CE applied after chick placement in growout houses (Goren et ah, 1988; E. Nurmi, 1989), because colonization of the chicks can occur before CE treatment. In an attempt to provide early protection of hatching chick against the hatchery contamination, CE treatment was applied prior to exposure of the newly hatched chick to salmonellae. The present study suggests that in ovo introduction of CE culture may be a feasible alternate route of applying
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on January 21, 2015
In Ovo Experiment 2. Experiment 2 was TABLE 1. Effect of in ovo competitive exclusion designed to evaluate the efficacy of CE culture treatment on hatchability and resistance to colonization of day-of-hatch chicks to a 10* culture administered in ovo to protect cell oral challenge of Salmonella typhimurium against S. typhimurium colonization. In the second experiment .1-mL quantities of Number chicks culture (diluted either 1:1,000 or 1: colonized/ 1,000,000) were injected either AC or BM on In ovo Percentage Number chicks Day 18 of incubation as previously treatment hatchability1 challenged described. On day of hatch, chicks were Uninjected (Control) 96a 11/12 orally gavaged with varying levels of AC 2 56 c ND3 Salmonella. Chicks from treated and un- AC(T)4 81b 0/4 78b 4/5 treated groups were evaluated 7 days AC(M)5 0d ND postchallenge for S. typhimurium coloniza- BM« 0d ND tion as previously described (Bailey et ah, BM(T)7 BM(M)8 44= 2/2 1988).
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COX ET AL.
TABLE 2. Resistance of day-of-hatch chicks to varying numbers of orally administered Salmonella typhitnurium cells following in ovo injection of a competitive exclusion culture into the air cell of embryonated eggs on the 18th day of incubation Number of salmonella cells administered 103
Trial
Untreated AC(T) 2 AC(M) 3
7/8 1 0/8 4/6 2 6/8 4/8 4/7 13/16 1 and 2 (81%)" 4/16 (25%)" 8/13 (62%)a>>
Untreated AC(T) AC(M) Untreated AC(T) AC(M)
1
10 s
107
4/4 0/7 7/8 6/8 4/6 2/8 10/12 (83%)a 4/13 (31%)b 9/16 (75%)a
6/6 4/8 7/8 7/7 5/6 6/6 13/13 (100%)a 9/14 (64%)a 13/14 (93%)a
a b
- Values within a column with no superscripts differ significantly (P < .05). dumber of chicks colonized/number challenged. 2 Air cell was the site of injection 48-h VL culture was diluted 1:1,000. 3 Air cell was the site of injection 48-h VL culture was diluted 1:1,000,000.
common of chicks and the and the
this treatment commercially. In some experiments, hatchability approached commercial acceptability and chicks demonstrated significant resistance to colonization by high challenge levels of S. typhimurium on day of hatch. ACKNOWLEDGMENTS
The authors wish to thank D. Posey, L. Tanner, T. Bryan, T. O'Connell, and L. Murray for their able technical assistance and J. Bandler, S. Smith, and E. Demesa for their able secretarial assistance.
REFERENCES 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. Barnes, E. M, G. C. Mead, D. A. Barnum, and E. G. Harry, 1972. The intestinal flora of the chicken in the period 2 to 6 weeks of age with particular reference to the anaerobic bacteria. Br. Poult. Sci. 13:311-326. Barnes, E. M., C. S. Impey, and D. M. Cooper, 1980.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on January 21, 2015
Treatment
Competitive exclusion of salmonellae from newly hatched chicks. Vet. Rec. 106(3):61. Blanchfield, B., M. A. Gardiner, and H. Pivnick, 1982. Nurmi concept for preventing infection of chicks by Salmonella: Comparison of fecal suspension and fecal cultures administered into the crop and in drinking water. J. Food Prot. 45: 345-347. Cox, N. A., J. S. Bailey, J. M. Mauldin, and L. C. Blankenship, 1990. Presence and impact of salmonellae 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. Goren, E., W. A. dejong, P. Doornenbal, N. M. Bolder, R.W.A.W. Mulder, and A. Jansen, 1988. Reduction of salmonella infection of broilers by spray application of intestinal microflora: a longitudinal study. Vet. Q. 10(4):249-255. Huttner, B., H. Landgraf, and E. Vielitz, 1981. Kontrolle der Salmonelleninfectionen in Mastelterntier-Bestanden durch Verabreichung von SPF-Darmflora on Eintagskuken. Dtsch. Tieraerztl. Wochenschr. 88:527-532. Nurmi, E., and M. Rantala, 1973. New aspects of salmonella infection in broiler production. Nature 241:210-211. Pivnick, H., B. Blanchfield, and J. Y. D'Aoust, 1981. Prevention of Salmonella infection in chicks by treatment with fecal cultures from mature chickens (Nurmi cultures). J. Food Prot. 44: 909-916. Pivnick, H., and E. Nurmi, 1982. The Nurmi concept and its role in the control of salmonellae in poultry. Chapter 2. Page 41 in: Developments in Food Microbiology. Volume 1. R. Davis, ed. Application of Science, Barking, England. Rantala, M., and E. Nurmi, 1973. Prevention of the growth of Salmonella infantis in chickens by flora of the alimentary tract of chickens. Br. Poult. Sci. 14:627-630. Seuna, E., 1979. Sensitivity of young chickens to Salmonella typhimurium var. Copenhagen and S. infantis infection and the preventive effect of cultured intestinal microflora. Avian Dis. 23: 392-400. Snoeyenbos, G. H„ O. M. Weinak, and C. F. Smyser, 1978. Protecting chicks and poultry from salmonellae by oral administration of "normal" gut microflora. Avian Dis. 22:273-287. Statistical Package for the Social Sciences, 1988. SPSS/PC+ V2.0 Base Manual for the IBM PC/ XT/AT and PS/2, Statistical Package for the Social Sciences, Chicago, IL. Soerjadi, A. S., S. M. Stehman, G. H. Snoeyenbos, O. M., Weinack, and C. F. Smyser, 1981. Some measurements of protection against Paratyphoid Salmonella and Escherichia coli by competitive exclusion in chickens. Avian Dis. 25: 706-712. Wierup, M., M. Wold-Troell, E. Nurmi, and M. Hakkinen, 1987. Epidemiological evaluation of the Salmonella controlling effect of a nationwide use of a "competitive exclusion" culture in poultry. Page 7 in: Proceeding of an International Workshop on Competitive Exclusion of Salmonellas from Poultry. Bristol Laboratory, Langford, UK.
ABSTRACT Exposure of chicks to salmonellae in the hatchery and hatchery environment limits the effectiveness of a competitive exclusion (CE) culture treatment. Therefore, in an attempt to apply treatment before chicks are exposed to salmonellae, the CE culture was introduced in ovo to unhatched embryos. An undefined, anaerobically grown CE culture, derived from cecal contents of healthy adult chickens, was diluted 1:1,000 or 1:1,000,000 and inoculated either into the air cell or beneath the inner air cell membrane of 17-day-old incubating hatching eggs. The treated chicks were more resistant than untreated chicks to varying challenge levels of Salmonella typhimurium, indicating that it may be possible to initiate protection of chicks to salmonellae challenge prior to hatching into a contaminated environment. (Key words: eggs, Salmonella, broiler chicks, colonization resistance, competitive exclusion) 1992 Poultry Science 71:1781-1784
sion (CE) or the Nurmi concept. Since the early 1970s, the efficacy of CE has been Research studies in Finland in the early demonstrated in a number of laboratories 1970s suggested that susceptibility of the (Snoeyenbos et at, 1978; Barnes et al, 1980; newly hatched broiler chick to salmonellae Pivnick et al., 1981; Bailey et al, 1988). colonization was due to delayed establish- Although the efficacy of CE has been ment of normal gut microflora in chicks clearly demonstrated in the laboratory, the reared according to modern mass produc- limited number of large-scale field trials tion methods (Nurmi and Rantala, 1973). that have been conducted have produced They also demonstrated that salmonellae mixed results (Huttner et al, 1981; Weirup infections could be prevented by feeding et al, 1987; Goren et al, 1988). anaerobically grown cultures obtained At hatch, most chicks have very little from the intestinal flora of adult fowl to microflora in their gut and are highly newly hatched chicks (Nurmi and Rantala, susceptible to intestinal colonization by 1973; Rantala and Nurmi, 1973). This salmonellae (Pivnick and Nurmi, 1982). By technique is known as competitive exclu- 2 wk of age, chicks have a mature gut microflora (Barnes et al, 1972) and are therefore much more resistant to intestinal colonization by salmonellae. Competitive Received for publication January 21, 1992. exclusion cultures given to chicks on the Accepted for publication June 19, 1992. day of hatch speed maturation of gut ! To whom correspondence should be addressed. 2 Embrex Inc., P.O. Box 13989, Research Triangle microflora and within a few hours significantly increase the resistance of chicks to Park, NC 27709. INTRODUCTION
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Embrex Inc., P.O. Box 13989, Research Triangle Park, North Carolina 27709
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COX ET AL.
salmonellae (Seuna, 1979; Soerjadi et al, 1981). Commercial hatchery environments have been shown to be significantly contaminated with salmonellae (Cox et al, 1990b, 1991), which can limit the effectiveness of CE treatment (Goren et al, 1988; E. Nurmi, National Veterinary Institute, Helsinki, Finland, personal communication, 1989). The objective of the present study was to assess the benefit of applying CE treatment prior to environmental exposure of chicks to salmonellae.
Competitive Exclusion Culture Fecal and cecal droppings were collected from 1-yr-old, salmonellae-free, caged layer chickens. These droppings (.5 g) were inoculated into 10 mL of Viande-Levure (VL) broth (Bailey et al, 1988) and incubated anaerobically at 35 C for 48 h. This culture (.2 mL) was subcultured into 10 mL of VL broth and similarly incubated anaerobically. Following incubation, glycerol was added to the broth at a final concentration of 15%. This culture with glycerol was divided into 1.0-mL portions, displaced into small plastic vials, and frozen at -70 C. For each experiment, a vial of the frozen culture was thawed and .2 mL of the culture was used to inoculate 10 mL of VL broth, which was then incubated for 48 h at 35 C. Salmonellae Chicks were challenged with a nalidixicacid-resistant strain of Salmonella typhimurium prepared according to the procedures of Bailey et al. (1988). Challenge levels were either 103, 1CP, or 107 per chick. In Ovo Experiments In ovo treatment with CE cultures was made directly onto the inner air cell shell membrane (AC) or beneath the inner membrane (BM) of 18-day-old incubating embryos. Three days post-treatment (Day 21 of incubation), control chicks and chicks treated on the day of hatch in each experiment were orally challenged by gavage with varying numbers of Salmonella
3
Sanofi Animal Health Inc., Berlin, MD 21811.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on January 21, 2015
MATERIALS AND METHODS
typhimurium cells. Seven days postchallenge, chicks were euthanatized by CO2 asphyxiation and then soaked in 70% ethyl alcohol for 1 min to reduce external bacterial contamination. Both ceca from each chick were then excised aseptically and analyzed according to the swab plate method described by Bailey et al. (1988) for recovery of the nalidixic acid marker resistant strain of S. typhimurium. In Ovo Experiment 1. This experiment was conducted as a pilot study. On Day 18 of incubation, 700 commercial broiler eggs containing live embryos, as determined by candling, were removed from an incubator and divided at random into seven groups. Untreated control eggs in the first group were transferred to a hatching basket. Eggs in the second group were injected with undiluted CE culture at the time of transfer from the setter to the hatcher. The shell was pierced at the pole of the air cell end of the egg with an 18-gauge hypodermic needle inserted to a depth of 2 to 5 mm past the shell surface. The 18-gauge needle was withdrawn and a syringe with a 22-gauge hypodermic needle was inserted into the hole in the shell to a depth of 2 to 5 mm. Also, 100 JUL of undiluted CE culture was injected onto the AC membrane. Eggs comprising the third and fourth treatment groups received 100 |iL of 1:1,000 or 1:1,000,000 diluted CE culture, respectively. Sanofi Marek's diluent3 was used to dilute the CE culture. The shells of the eggs in the remaining groups were pierced as described above, but during transfer were injected with a 22-gauge needle to a depth of 20 to 25 mm beneath the shell in order to deliver the CE culture BM. Eggs in the fifth, sixth, and seventh treatment groups were injected with 100 uL of undiluted CE culture, 1:1,000 diluted CE culture, and 1: 1,000,000 diluted CE culture, respectively. Eggs were returned to the incubator for 3 additional days of incubation. Hatchability of each group was noted and expressed as the percentage of live chicks. Hatched chicks in the control and several treatment groups were orally challenged with 106 S. typhimurium cells and evaluated for resistance to colonization as previously described.
RESEARCH NOTE
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a_d
Statistical Analyses Frequency data (including hatchability) from each experiment were subjected to chisquare analysis (Statistical Package for the Social Sciences, 1988). In Experiment 2, frequency data were pooled across replicates and the pooled data were analyzed. A significance level of P < .05 was selected. RESULTS AND DISCUSSION When varying dilutions of anaerobically grown, CE culture was administered in ovo in the AC or BM, only the 1:1,000 and 1:1,000,000 dilutions administered into the air cell resulted in hatchabilities approaching commercial acceptability (Table 1). Only the chicks inoculated in ovo onto the air cell with a 1:1,000 dilution of the CE culture acquired resistance to a 106 cell oral challenge of S. typhimurium on the day of hatch when compared with the untreated controls. Because the number of chicks in the first experiment was limited, a second series of experiments was conducted utilizing a greater number of day-of-hatch chicks with varying Salmonella challenge dosages (Table 2). In Trial 1 of these studies, in ovo introduction of a 1:1,000 dilution of CE culture onto AC of Day 18 embryonated eggs produced day-of-hatch chicks completely resistant to 103 and 105 S. typhimurium challenge. In addition, only four of eight chicks treated with CE culture were colonized when challenged with 107 S. typhimurium, whereas all of the untreated chicks were colonized. The colonization resistance to challenge was less evident in Trial 2 than in Trial 1.
Values in a column with no common superscripts differ significantly (P < .05). !Number of normal chicks hatched out of 100 fertile eggs set. 2 The air cell was the site of injection with undiluted 48-h VL culture. 3 ND = not done. 4 Same as (2) except that the VL culture was diluted 1:1,000. 'Same as (2) except that the VL culture was diluted 1:1,000,000. 6 Injection was beneath the inner air cell membrane of the egg with undiluted culture. 7 Same as (6) except that the VL culture was diluted 1:1,000. 8 Same as (6) except that the VL culture was diluted 1:1,000,000.
Pooled data revealed significantly different resistances among treated chicks to challenges of S. typhimurium after in ovo injection of a 1:1,000 dilution. Competitive exclusion has been demonstrated in laboratories in various countries to be an effective method of reducing salmonellae colonization in the intestinal tract of chickens (Snoeyenbos et ah, 1978; Blanchfield et ah, 1982; Bailey et ah, 1988). The presence and persistence of salmonellae in commercial hatcheries (Cox et ah, 1990, 1991) limits the effectiveness of CE applied after chick placement in growout houses (Goren et ah, 1988; E. Nurmi, 1989), because colonization of the chicks can occur before CE treatment. In an attempt to provide early protection of hatching chick against the hatchery contamination, CE treatment was applied prior to exposure of the newly hatched chick to salmonellae. The present study suggests that in ovo introduction of CE culture may be a feasible alternate route of applying
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on January 21, 2015
In Ovo Experiment 2. Experiment 2 was TABLE 1. Effect of in ovo competitive exclusion designed to evaluate the efficacy of CE culture treatment on hatchability and resistance to colonization of day-of-hatch chicks to a 10* culture administered in ovo to protect cell oral challenge of Salmonella typhimurium against S. typhimurium colonization. In the second experiment .1-mL quantities of Number chicks culture (diluted either 1:1,000 or 1: colonized/ 1,000,000) were injected either AC or BM on In ovo Percentage Number chicks Day 18 of incubation as previously treatment hatchability1 challenged described. On day of hatch, chicks were Uninjected (Control) 96a 11/12 orally gavaged with varying levels of AC 2 56 c ND3 Salmonella. Chicks from treated and un- AC(T)4 81b 0/4 78b 4/5 treated groups were evaluated 7 days AC(M)5 0d ND postchallenge for S. typhimurium coloniza- BM« 0d ND tion as previously described (Bailey et ah, BM(T)7 BM(M)8 44= 2/2 1988).
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TABLE 2. Resistance of day-of-hatch chicks to varying numbers of orally administered Salmonella typhitnurium cells following in ovo injection of a competitive exclusion culture into the air cell of embryonated eggs on the 18th day of incubation Number of salmonella cells administered 103
Trial
Untreated AC(T) 2 AC(M) 3
7/8 1 0/8 4/6 2 6/8 4/8 4/7 13/16 1 and 2 (81%)" 4/16 (25%)" 8/13 (62%)a>>
Untreated AC(T) AC(M) Untreated AC(T) AC(M)
1
10 s
107
4/4 0/7 7/8 6/8 4/6 2/8 10/12 (83%)a 4/13 (31%)b 9/16 (75%)a
6/6 4/8 7/8 7/7 5/6 6/6 13/13 (100%)a 9/14 (64%)a 13/14 (93%)a
a b
- Values within a column with no superscripts differ significantly (P < .05). dumber of chicks colonized/number challenged. 2 Air cell was the site of injection 48-h VL culture was diluted 1:1,000. 3 Air cell was the site of injection 48-h VL culture was diluted 1:1,000,000.
common of chicks and the and the
this treatment commercially. In some experiments, hatchability approached commercial acceptability and chicks demonstrated significant resistance to colonization by high challenge levels of S. typhimurium on day of hatch. ACKNOWLEDGMENTS
The authors wish to thank D. Posey, L. Tanner, T. Bryan, T. O'Connell, and L. Murray for their able technical assistance and J. Bandler, S. Smith, and E. Demesa for their able secretarial assistance.
REFERENCES 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. Barnes, E. M, G. C. Mead, D. A. Barnum, and E. G. Harry, 1972. The intestinal flora of the chicken in the period 2 to 6 weeks of age with particular reference to the anaerobic bacteria. Br. Poult. Sci. 13:311-326. Barnes, E. M., C. S. Impey, and D. M. Cooper, 1980.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on January 21, 2015
Treatment
Competitive exclusion of salmonellae from newly hatched chicks. Vet. Rec. 106(3):61. Blanchfield, B., M. A. Gardiner, and H. Pivnick, 1982. Nurmi concept for preventing infection of chicks by Salmonella: Comparison of fecal suspension and fecal cultures administered into the crop and in drinking water. J. Food Prot. 45: 345-347. Cox, N. A., J. S. Bailey, J. M. Mauldin, and L. C. Blankenship, 1990. Presence and impact of salmonellae 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. Goren, E., W. A. dejong, P. Doornenbal, N. M. Bolder, R.W.A.W. Mulder, and A. Jansen, 1988. Reduction of salmonella infection of broilers by spray application of intestinal microflora: a longitudinal study. Vet. Q. 10(4):249-255. Huttner, B., H. Landgraf, and E. Vielitz, 1981. Kontrolle der Salmonelleninfectionen in Mastelterntier-Bestanden durch Verabreichung von SPF-Darmflora on Eintagskuken. Dtsch. Tieraerztl. Wochenschr. 88:527-532. Nurmi, E., and M. Rantala, 1973. New aspects of salmonella infection in broiler production. Nature 241:210-211. Pivnick, H., B. Blanchfield, and J. Y. D'Aoust, 1981. Prevention of Salmonella infection in chicks by treatment with fecal cultures from mature chickens (Nurmi cultures). J. Food Prot. 44: 909-916. Pivnick, H., and E. Nurmi, 1982. The Nurmi concept and its role in the control of salmonellae in poultry. Chapter 2. Page 41 in: Developments in Food Microbiology. Volume 1. R. Davis, ed. Application of Science, Barking, England. Rantala, M., and E. Nurmi, 1973. Prevention of the growth of Salmonella infantis in chickens by flora of the alimentary tract of chickens. Br. Poult. Sci. 14:627-630. Seuna, E., 1979. Sensitivity of young chickens to Salmonella typhimurium var. Copenhagen and S. infantis infection and the preventive effect of cultured intestinal microflora. Avian Dis. 23: 392-400. Snoeyenbos, G. H„ O. M. Weinak, and C. F. Smyser, 1978. Protecting chicks and poultry from salmonellae by oral administration of "normal" gut microflora. Avian Dis. 22:273-287. Statistical Package for the Social Sciences, 1988. SPSS/PC+ V2.0 Base Manual for the IBM PC/ XT/AT and PS/2, Statistical Package for the Social Sciences, Chicago, IL. Soerjadi, A. S., S. M. Stehman, G. H. Snoeyenbos, O. M., Weinack, and C. F. Smyser, 1981. Some measurements of protection against Paratyphoid Salmonella and Escherichia coli by competitive exclusion in chickens. Avian Dis. 25: 706-712. Wierup, M., M. Wold-Troell, E. Nurmi, and M. Hakkinen, 1987. Epidemiological evaluation of the Salmonella controlling effect of a nationwide use of a "competitive exclusion" culture in poultry. Page 7 in: Proceeding of an International Workshop on Competitive Exclusion of Salmonellas from Poultry. Bristol Laboratory, Langford, UK.
