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Research Note: Extent of Salmonellae Contamination in Breeder Hatcheries
Research Note: Extent of Sahnonellae Contamination in Breeder Hatcheries N. A. COX,1 J. S. BAILEY,1 J. M. MAULDIN,2 L. C. BLANKENSHIP,1 and J. L. WILSON2 USDA, Agricultural Research Service, Russell Research Center, P.O. Box 5677, Athens, Georgia 30613 and Extension Poultry Science Department, The University of Georgia, Athens, Georgia 30602 (Received for publication September 5, 1990) ABSTRACT Egg fragments, paper pads from chick boxes, and fluff samples were obtained from six commercial broiler breeder hatcheries and analyzed for the presence and level of sahnonellae. Overall, 42 of 380 samples (11.1%) from those hatcheries were contaminated with sahnonellae. Sahnonellae organisms were detected in 22 of 145 (152%), 5 of 100 (4.6%), and 15 of 125 (12%) samples of egg fragments, fluff, and paper pads, respectively. The percentage salmonellae-positive samples from each of the six hatcheries were 1.3, 5.0, 22.5, 11.4, 36.0, and 4.3% respectively. Of the 140 samples randomly selected for enumeration, sahnonellae were found in 11 samples. Four of these 11 samples had greater than 10 3 sahnonellae per sample, 3 others had greater than 102 but less man 103, and the remaining 4 had less than 102. Salmonella serotypes isolated were S. berta, S. California, S. give, S. hadar, S. mbandaka, S. senftenberg, and S. typhimurium, all of which have previously been isolated from poultry. The incidence and extent of salmonellae-positive samples found in the breeder hatcheries were much less than that previously found in broiler hatcheries. Many factors contribute to the lower incidence and level of sahnonellae found in the breeder hatcheries; however bom the breeder and broiler hatcheries present critical control points in the prevention of sahnonellae contamination during commercial poultry production. The cycle of sahnonellae contamination will not likely be broken until contamination at these critical points is eliminated. (Key words: broiler breeder hatcheries, Salmonella, frequency of isolation, chick boxes, egg fragments) 1991 Poultry Science 70:416-418 INTRODUCTION
Fertile eggs leaving die breeder house could carry many bacteria, both those on the shell surface and others that have penetrated beneath the shell. Dirty nesting material can contribute to egg contamination. In addition to surface contamination, a freshly laid egg that is wet and warm is susceptible to rapid penetration by microorganisms, and these contaminated eggs have the potential for spreading sahnonellae in the hatchery (Williams and Dillard, 1968). Many studies have demonstrated that the Salmonella serotypes isolated from hatchery samples can subsequendy be isolated from die grow out houses, and on carcasses after processing (Bhatia and McNabb, 1980; Lahellec and Colin, 1985; Goren et al., 1988). Favorable conditions (i.e., temperature and humidity) for microbial growth make the hatchery an initial point for die propagation
Russell Research Center. University of Georgia.
and proliferation of bacteria (including sahnonellae) in me broiler production process. Contamination of embryos can occur in die incubator but probably more frequendy occurs in die hatcher during pipping and hatching and during transportation to die farm. In a previous study of commercial broiler hatcheries, 75% of 175 samples were salmonellae-positive, and 38 of 40 samples enumerated contained greater man 10 3 sahnonellae (Cox et al., 1990). The objectives of die present study were to determine die incidence, extent, and serotypes of Salmonella in broiler breeder hatcheries. MATERIALS AND METHODS
In die present study, all of die egg sources were from primary breeder flocks except for Hatchery 5, whose egg source was a great grandparent breeder flock. Egg fragments, fluff, and paper pads were obtained from each hatchery on each of two sampling days, witii one exception: Hatchery 5 was sampled on only one visit Randomly selected samples
416
417
RESEARCH NOTE TABLE 1. Presence of salmonellae contamination in broiler breeder hatcheries Material sampled Hatchery
Egg fragments
Fluff
Paper pads
Total
1 2 3 4 5 6 Total
0/351 3/20 3/20 6/30 9/10 1/30 22/145 (15.2%)
0/20 0/20 1/20 2/20 0/10 2/20 5/110 (4.5%)
1/20 0/20 14/40 0/20 0/5 0/20 15/125 (12.0%)
1/75 (1.3%) 3/60 (5.0%) 18/80 (22.5%) 8/70 (11.4%) 9/25 (36.0%) 3/70 (4.3%) 42/380 (11.1%)
'Number of salmonellae-positive samples per number of samples tested.
(140) were semi-quantitatively analyzed by removing l/100th and l/l,000th of the buffered peptone (BP) prior to incubation, incubated separately, and then analyzed for the presence of salmonellae as described below. Approximately 10 g of egg fragments were aseptically removed from the hatching trays and placed in a sterile plastic bag containing 90 mL of BP medium (Juven et al., 1984). Approximately 5 g of chick fluff were aseptically removed from the hatching cabinet and placed in a sterile plastic bag containing SO mL of BP. Each paper pad was cut into pieces using sterile scissors and placed in a sterile plastic bag containing 500 mL of BP. Following overnight incubation of BP at 37 C, 1 mL of the BP was aseptically transferred to 9 mL of tetrathionate broth (Difco;3 Hajna and Damon, 1956) and incubated 24 h at 43 C. Plates of brilliant green Sulfa3 and USDAMLIA4 (Bailey et al., 1988) were streaked for isolation with a loopful (3 mm loop) of TT broth. Following incubation of the plates for 24 to 48 h, two suspect colonies appearing on the plates were selected, biochemically screened (Lysine Iron Agar slants and MicroID),5 and then serologically confirmed to be Salmonella. Confirmed isolates were sent to the Diagnostics Bacteriology Laboratory, National Animal Disease Center, Ames, IA for final confirmation and determination of the serotype.
•'Difco Laboratories, Detroit, MI 48232-7058. 'HjSDA-MLIA United States Department of Agriculture-Modified Lysine Iron Agar. 5 Organon Teknika, Durham, NC 27704.
RESULTS
The percentage of salmonellae-positive samples obtained from six different breeder hatcheries ranged from as low as 1.3% (1 of 75) to as high as 36% (9 of 25) with the overall percent positive being 11.1% (42 out of 380) (Table 1). Salmonellae organisms were recovered from 22 of 145 (15.2%), 5 of 100 (4.6%), and 15 of 125 (12.0%) samples of egg fragments, fluff and paper pads, respectively. The Salmonella serotypes encountered in these samples were S. berta, S. California, S. give, S. hadar, S. mbandaka, S. senftenberg, and S. typhimurium, all of which have previously been isolated from poultry. In one of the hatcheries (Hatchery 3), three different serotypes were detected, while in other hatcheries only one serotype was found in each. There were 11 salmonellae-positive samples from the 140 samples randomly selected from the enumeration. Four of 11 had greater than 103 salmonellae per sample, three others had greater than 102 but less than 103, and the remaining four samples had less than 102. DISCUSSION
The incidence and extent of salmonellae found in the present study with broiler breeder hatcheries was much lower than that reported in a previous study with broiler hatcheries (Cox et al., 1990). There are many reasons for the lower incidence of salmonellae-positive samples in breeder versus broiler hatcheries. Typically, primary breeder flocks are smaller in size and more remotely located with very few visitors. The hatching eggs are gathered more frequently because of the size of the flocks and the value of the eggs. The eggs are often chemically disinfected shortly after gathering.
418
COX ET AL.
According to Wilson and Mauldin (1989), die following eight elements are crucial to a hatchery sanitation program: hatchery design, ventilation, isolation, disinfection, clean-up, waste handling, microbiological monitoring, and communication. Due to the increase value of each chick produced, the breeder hatcheries have state-of-the-art design and equipment. Ventilation systems filter incoming air, provide optimum air flow to dilute contaminants, and channel air from dirty areas in the hatchery outside the building. The ideal hatchery design allows one-way movement (clean to dirty) of materials and people. This flow pattern reduces the opportunity for contamination of clean areas. The reduced volume allows for a more intensive clean-up and disinfection program. Biosecurity of the breeder hatcheries is strictly adhered to, requiring shower-in procedures for workers and visitors. All of these mentioned factors can and do affect microbiological contamination, including salmonellae. Although materials sampled at the hatchery have been shown to be most useful for monitoring hatchery sanitation (Wright, 1958; Wright and Epps, 1958) and a useful indicator of flock infection (Bhatia and McNabb, 1980), others have suggested that these hatchery samples underestimate the salmonellae infection of the flocks. Snoeyenbos et al. (1970) were unable to isolate salmonellae from fluff of 18 hatches, in which hatching eggs were obtained from salmonella-infected breeding chickens. Also, Morris et al. (1969) reported that hatchery samples showed much lower frequency of salmonellae isolation compared with samples from flocks of breeders and broilers. Whether or not breeder hatchery samples underestimate salmonellae infection of breeder flocks, the present study clearly indicates that salmonellae are present in significant numbers in breeder hatcheries and that breeder flocks are early critical points for preventing salmonellae entry into the integrated poultry operation. In these particular hatcheries sampled, egg fragments and paper pads appear to be more significant sources of salmonellae contamination than fluff. One interesting observation was that Hatchery 1, the hatchery with the lowest-incidence of salmonellae-positive samples (1 out of 75) obtained eggs from caged primary breeders,
whereas the other five hatcheries obtained eggs from floor-raised breeders. It is unknown to what extent cage versus floor raising of breeding flocks can affect salmonellae contamination in a field study. ACKNOWLEDGMENTS
The authors wish to thank D. Posey, L. Tanner, and S. Larkin for their technical assistance and J. Bandler, S. Smith, and Elisa Demesa for the secretarial assistance. REFERENCES Bailey, J. S., J. Y. Chin, N. A. Cox, and R. W. Johnston, 1988. Improved selective procedure for detection of salmonellae from poultry and sausage products. J. Food Prot 51:391-396. Bhatia, T.R.S., and G. D. McNabb, 1980. Dissemination of Salmonella in broiler chicken operations. Avian Dis. 24:616-624. Cox, N. A., J. S. Bailey, J. M. Mauldin, and L. C. Blankenship, 1990. Research note: presence and impact of salmonellae contamination in commercial broiler hatcheries. Poultry Sci. 69:1606-1609. Goren, E., W. A. DeJong, P. Doorenbal, N. M. Bolder, R.WA.W. Mulder, and A. Jansen, 1988. Reduction of salmonellae infection of broilers by spray application of intestinal microflora; a longitudinal study. Vet. Q. 10:249-255. Hajna, A. A., and S. R. Damon, 1956. New enrichment and plating media for the isolation of Salmonella and Shigella organisms. Appl. Microbiol. 4:341-345. Juven, B. J., N. A. Cox, J. S. Bailey, and J. E. Thomson, 1984. Minimal medium recovery of Salmonella from dry feed. J. Food Prot 47:299-302. Lahellec, C , and P. Colin, 1985. Relationship between serotype of salmonellae from hatcheries and rearing farms and those from processed poultry carcasses. Br. Poult Sci. 26:179-186. Morris, G., B. McMurray, M. Galton, and J. Wells, 1969. A study of the dissemination of salmonellosis in a commercial broiler chicken operation. Am. J. Vet Res. 30:1413-1421. Snoeyenbos, G. H., B. A. Mackie, C. F. Smyser, and C. R. Weston, 1970. Progress in identifying and maintaining salmonellae-free commercial chicken breeding flocks. Avian Dis. 14:683-696. Williams, J. E., and L. H. DiHard, 1968. Salmonella penetration of fertile and infertile chicken eggs at progressive stages of inoculation. Avian Dis. 12: 629-635. Wilson, J. L., and J. M. Mauldin, 1989. Hatchery sanitation procedures after new formaldehyde use rules. Poult Dig. 48:406-407, 410. Wright, M. L., 1958. Hatchery sanitation. Can. J. Comp. Med. 22:62-69. Wright M- L., and N. A. Epps. 1958. Hatchery sanitation. Can. J. Comp. Med. 22:396-401.
Fertile eggs leaving die breeder house could carry many bacteria, both those on the shell surface and others that have penetrated beneath the shell. Dirty nesting material can contribute to egg contamination. In addition to surface contamination, a freshly laid egg that is wet and warm is susceptible to rapid penetration by microorganisms, and these contaminated eggs have the potential for spreading sahnonellae in the hatchery (Williams and Dillard, 1968). Many studies have demonstrated that the Salmonella serotypes isolated from hatchery samples can subsequendy be isolated from die grow out houses, and on carcasses after processing (Bhatia and McNabb, 1980; Lahellec and Colin, 1985; Goren et al., 1988). Favorable conditions (i.e., temperature and humidity) for microbial growth make the hatchery an initial point for die propagation
Russell Research Center. University of Georgia.
and proliferation of bacteria (including sahnonellae) in me broiler production process. Contamination of embryos can occur in die incubator but probably more frequendy occurs in die hatcher during pipping and hatching and during transportation to die farm. In a previous study of commercial broiler hatcheries, 75% of 175 samples were salmonellae-positive, and 38 of 40 samples enumerated contained greater man 10 3 sahnonellae (Cox et al., 1990). The objectives of die present study were to determine die incidence, extent, and serotypes of Salmonella in broiler breeder hatcheries. MATERIALS AND METHODS
In die present study, all of die egg sources were from primary breeder flocks except for Hatchery 5, whose egg source was a great grandparent breeder flock. Egg fragments, fluff, and paper pads were obtained from each hatchery on each of two sampling days, witii one exception: Hatchery 5 was sampled on only one visit Randomly selected samples
416
417
RESEARCH NOTE TABLE 1. Presence of salmonellae contamination in broiler breeder hatcheries Material sampled Hatchery
Egg fragments
Fluff
Paper pads
Total
1 2 3 4 5 6 Total
0/351 3/20 3/20 6/30 9/10 1/30 22/145 (15.2%)
0/20 0/20 1/20 2/20 0/10 2/20 5/110 (4.5%)
1/20 0/20 14/40 0/20 0/5 0/20 15/125 (12.0%)
1/75 (1.3%) 3/60 (5.0%) 18/80 (22.5%) 8/70 (11.4%) 9/25 (36.0%) 3/70 (4.3%) 42/380 (11.1%)
'Number of salmonellae-positive samples per number of samples tested.
(140) were semi-quantitatively analyzed by removing l/100th and l/l,000th of the buffered peptone (BP) prior to incubation, incubated separately, and then analyzed for the presence of salmonellae as described below. Approximately 10 g of egg fragments were aseptically removed from the hatching trays and placed in a sterile plastic bag containing 90 mL of BP medium (Juven et al., 1984). Approximately 5 g of chick fluff were aseptically removed from the hatching cabinet and placed in a sterile plastic bag containing SO mL of BP. Each paper pad was cut into pieces using sterile scissors and placed in a sterile plastic bag containing 500 mL of BP. Following overnight incubation of BP at 37 C, 1 mL of the BP was aseptically transferred to 9 mL of tetrathionate broth (Difco;3 Hajna and Damon, 1956) and incubated 24 h at 43 C. Plates of brilliant green Sulfa3 and USDAMLIA4 (Bailey et al., 1988) were streaked for isolation with a loopful (3 mm loop) of TT broth. Following incubation of the plates for 24 to 48 h, two suspect colonies appearing on the plates were selected, biochemically screened (Lysine Iron Agar slants and MicroID),5 and then serologically confirmed to be Salmonella. Confirmed isolates were sent to the Diagnostics Bacteriology Laboratory, National Animal Disease Center, Ames, IA for final confirmation and determination of the serotype.
•'Difco Laboratories, Detroit, MI 48232-7058. 'HjSDA-MLIA United States Department of Agriculture-Modified Lysine Iron Agar. 5 Organon Teknika, Durham, NC 27704.
RESULTS
The percentage of salmonellae-positive samples obtained from six different breeder hatcheries ranged from as low as 1.3% (1 of 75) to as high as 36% (9 of 25) with the overall percent positive being 11.1% (42 out of 380) (Table 1). Salmonellae organisms were recovered from 22 of 145 (15.2%), 5 of 100 (4.6%), and 15 of 125 (12.0%) samples of egg fragments, fluff and paper pads, respectively. The Salmonella serotypes encountered in these samples were S. berta, S. California, S. give, S. hadar, S. mbandaka, S. senftenberg, and S. typhimurium, all of which have previously been isolated from poultry. In one of the hatcheries (Hatchery 3), three different serotypes were detected, while in other hatcheries only one serotype was found in each. There were 11 salmonellae-positive samples from the 140 samples randomly selected from the enumeration. Four of 11 had greater than 103 salmonellae per sample, three others had greater than 102 but less than 103, and the remaining four samples had less than 102. DISCUSSION
The incidence and extent of salmonellae found in the present study with broiler breeder hatcheries was much lower than that reported in a previous study with broiler hatcheries (Cox et al., 1990). There are many reasons for the lower incidence of salmonellae-positive samples in breeder versus broiler hatcheries. Typically, primary breeder flocks are smaller in size and more remotely located with very few visitors. The hatching eggs are gathered more frequently because of the size of the flocks and the value of the eggs. The eggs are often chemically disinfected shortly after gathering.
418
COX ET AL.
According to Wilson and Mauldin (1989), die following eight elements are crucial to a hatchery sanitation program: hatchery design, ventilation, isolation, disinfection, clean-up, waste handling, microbiological monitoring, and communication. Due to the increase value of each chick produced, the breeder hatcheries have state-of-the-art design and equipment. Ventilation systems filter incoming air, provide optimum air flow to dilute contaminants, and channel air from dirty areas in the hatchery outside the building. The ideal hatchery design allows one-way movement (clean to dirty) of materials and people. This flow pattern reduces the opportunity for contamination of clean areas. The reduced volume allows for a more intensive clean-up and disinfection program. Biosecurity of the breeder hatcheries is strictly adhered to, requiring shower-in procedures for workers and visitors. All of these mentioned factors can and do affect microbiological contamination, including salmonellae. Although materials sampled at the hatchery have been shown to be most useful for monitoring hatchery sanitation (Wright, 1958; Wright and Epps, 1958) and a useful indicator of flock infection (Bhatia and McNabb, 1980), others have suggested that these hatchery samples underestimate the salmonellae infection of the flocks. Snoeyenbos et al. (1970) were unable to isolate salmonellae from fluff of 18 hatches, in which hatching eggs were obtained from salmonella-infected breeding chickens. Also, Morris et al. (1969) reported that hatchery samples showed much lower frequency of salmonellae isolation compared with samples from flocks of breeders and broilers. Whether or not breeder hatchery samples underestimate salmonellae infection of breeder flocks, the present study clearly indicates that salmonellae are present in significant numbers in breeder hatcheries and that breeder flocks are early critical points for preventing salmonellae entry into the integrated poultry operation. In these particular hatcheries sampled, egg fragments and paper pads appear to be more significant sources of salmonellae contamination than fluff. One interesting observation was that Hatchery 1, the hatchery with the lowest-incidence of salmonellae-positive samples (1 out of 75) obtained eggs from caged primary breeders,
whereas the other five hatcheries obtained eggs from floor-raised breeders. It is unknown to what extent cage versus floor raising of breeding flocks can affect salmonellae contamination in a field study. ACKNOWLEDGMENTS
The authors wish to thank D. Posey, L. Tanner, and S. Larkin for their technical assistance and J. Bandler, S. Smith, and Elisa Demesa for the secretarial assistance. REFERENCES Bailey, J. S., J. Y. Chin, N. A. Cox, and R. W. Johnston, 1988. Improved selective procedure for detection of salmonellae from poultry and sausage products. J. Food Prot 51:391-396. Bhatia, T.R.S., and G. D. McNabb, 1980. Dissemination of Salmonella in broiler chicken operations. Avian Dis. 24:616-624. Cox, N. A., J. S. Bailey, J. M. Mauldin, and L. C. Blankenship, 1990. Research note: presence and impact of salmonellae contamination in commercial broiler hatcheries. Poultry Sci. 69:1606-1609. Goren, E., W. A. DeJong, P. Doorenbal, N. M. Bolder, R.WA.W. Mulder, and A. Jansen, 1988. Reduction of salmonellae infection of broilers by spray application of intestinal microflora; a longitudinal study. Vet. Q. 10:249-255. Hajna, A. A., and S. R. Damon, 1956. New enrichment and plating media for the isolation of Salmonella and Shigella organisms. Appl. Microbiol. 4:341-345. Juven, B. J., N. A. Cox, J. S. Bailey, and J. E. Thomson, 1984. Minimal medium recovery of Salmonella from dry feed. J. Food Prot 47:299-302. Lahellec, C , and P. Colin, 1985. Relationship between serotype of salmonellae from hatcheries and rearing farms and those from processed poultry carcasses. Br. Poult Sci. 26:179-186. Morris, G., B. McMurray, M. Galton, and J. Wells, 1969. A study of the dissemination of salmonellosis in a commercial broiler chicken operation. Am. J. Vet Res. 30:1413-1421. Snoeyenbos, G. H., B. A. Mackie, C. F. Smyser, and C. R. Weston, 1970. Progress in identifying and maintaining salmonellae-free commercial chicken breeding flocks. Avian Dis. 14:683-696. Williams, J. E., and L. H. DiHard, 1968. Salmonella penetration of fertile and infertile chicken eggs at progressive stages of inoculation. Avian Dis. 12: 629-635. Wilson, J. L., and J. M. Mauldin, 1989. Hatchery sanitation procedures after new formaldehyde use rules. Poult Dig. 48:406-407, 410. Wright, M. L., 1958. Hatchery sanitation. Can. J. Comp. Med. 22:62-69. Wright M- L., and N. A. Epps. 1958. Hatchery sanitation. Can. J. Comp. Med. 22:396-401.