Cross Contamination of Turkey Carcasses by Salmonella Species During Defeathering1

Cross Contamination of Turkey Carcasses by Salmonella Species During Defeathering1 C. W. Nde, J. M. McEvoy, J. S. Sherwood, and C. M. Logue2 The Great Plains Institute of Food Safety, Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo 58105 increased after defeathering during visits 1 and 4. Over the 4 visits, all Salmonella subtypes obtained after defeathering were also isolated before defeathering. The results of this study suggest that Salmonella was transferred from the feathers to carcass skin during each visit. On each visit, the Salmonella subtypes isolated from the fingers of the picker machines were similar to subtypes isolated before and after defeathering, indicating that the fingers facilitate carcass cross contamination during defeathering. Salmonella isolated from scald water during visit 4 was related to isolates obtained before and after defeathering, suggesting that scald water is also a vehicle for cross contamination during defeathering. By using molecular subtyping, this study demonstrated the relationship between Salmonella present on the feathers of live turkeys and carcass skin after defeathering, suggesting that decontamination procedures applied to the external surfaces of live turkeys could reduce Salmonella cross contamination during defeathering.

Key words: Salmonella, turkey, defeathering, cross contamination 2007 Poultry Science 86:162–167

human salmonellosis. To date, most studies of Salmonella cross contamination during poultry processing have used broilers, and there is limited information relating to turkeys. Birds presented for processing may harbor Salmonella in their feces but not show any symptoms of disease (Rigby and Petit, 1980, 1981; Higgins et al., 1982) and serve as potential contamination sources to uncontaminated birds. Defeathering, evisceration, and chilling are processing stages where cross contamination may occur (James et al., 1992; Hafez et al., 1997; Ono and Yamamoto, 1999). Contaminated external surfaces such as feathers may have a direct contaminating effect on carcass skin during early processing stages, but there are few data documenting this effect. To date, no study has examined the relationship between Salmonella contamination of the feathers of live birds and defeathered carcasses. The defeathering process, which consists of scalding, followed by mechanical feather removal, has been reported to be a site of significant microbial cross contami-

INTRODUCTION Salmonella is one of the leading causes of bacterial foodborne illness in the United States, with an annual economic impact estimated at $0.5 to 2.3 billion (Frenzen et al., 1999). Poultry meat is commonly implicated in human salmonellosis (Morris and Wells, 1970), with broilers and turkeys reported as vehicles. Identifying sources of contamination during slaughter and processing and implementing strategies to reduce, eliminate, or prevent such contamination are key to reducing the prevalence of Salmonella on poultry and the consequent incidence of

2007 Poultry Science Association Inc. Received May 10, 2006. Accepted September 30, 2006. 1 Acknowledgement of grant: USDA CSREES Special Research Grant # TE 2003-06068. A preliminary report of this study was published in the proceedings of the Conference of Research Workers in Animal Disease, Chicago, IL, November 2004. 2 Corresponding author: [email protected]

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ABSTRACT Salmonella present on the feathers of live birds could be a source of contamination to carcass skin during defeathering. In this study, the possibility of transfer of Salmonella from the feathers of live turkeys to carcass tissue during the defeathering process at a commercial turkey processing plant was investigated. The contribution of scald water and the fingers of the picker machines to cross contamination were also examined. Over 4 visits, swab samples were collected from 174 randomly selected tagged birds before and after defeathering. Two swab samples from the fingers of the picker machines and a sample of scald water were also collected during each visit. Detection of Salmonella was carried out following standard cultural and identification methods. The DNA fingerprints obtained from pulsed field gel electrophoresis of Salmonella serotypes isolated before and after defeathering, from scald water, and from the fingers of the picker machines were compared to trace cross contamination routes. Salmonella prevalence was similar before and after defeathering during visits 2 and 3 and significantly

SALMONELLA CROSS CONTAMINATION DURING TURKEY DEFEATHERING

MATERIALS AND METHODS Description of the Defeathering System This study was carried out in a commercial turkey processing plant that processes live turkeys to raw and cooked finished products. Turkeys were processed at an average speed of 800 carcasses/h. Birds were stunned, killed, and exsanguinated as they were navigated through a 20 to 30 ft tunnel. Carcasses were immersed in a scald tank that was continuously supplied with steam-heated water pumped through pipes at the bottom of the tanks. Samples were collected on each of 4 visits to the plant that were planned to coincide with the slaughter of flocks from farms with a history of Salmonella contamination. The temperature of scald tank water on the first 2 study days was 60°C and was 63.5°C during the third and fourth visits. The average immersion time per bird in the scald tank was 2.2 min. After scalding, the carcasses were conveyed to the picker machines, which consisted of 2 compartments. The first compartment was a Barker Gent-LFlex picker with hock and straddle picker add-ons (Barker/Foodcraft, New Holland, PA). This compartment removes about 90% of all feathers. The second compartment was a Dura II-T (Simon Johnson, Barker/Foodcraft)

picker, which removes the remaining feathers. The total time for feather removal for each carcass was about 2.2 min, bringing the total defeathering time to 4.4 min.

Sample Collection To ensure that the same randomly selected birds were sampled before and after the defeathering process during each visit, brightly colored plastic zip-ties were interlocked on the metal link above the shackles holding the selected live birds. After the tagged carcasses emerged from the picker machine, a second set of samples was collected. Over 4 visits, 174 samples were collected at predefeathering and 174 samples were collected at postdefeathering. Two swab samples were collected from the fingers of the picker machines, and 1 sample of scald water was collected during each visit.

Sampling Method Commercially available sponges (Whirl-Pak SpeciSponge, Nasco, Fort Atkinson, WI) moistened with 10mL single strength buffered peptone water (BPW; Oxoid Ltd., Basingstoke, UK) were used for carcass and picker finger sampling. For carcass sampling prior to defeathering, using a sterile gloved hand, the sponge was squeezed inside the bag to remove excess liquid before using it to swab an undelimited 100 cm2 area on the breast feathers of randomly selected tagged live birds. Following defeathering, an undelimited 100 cm2 area of the carcass breast tissue of the tagged defeathered bird was swabbed. Swabbing before and after defeathering was carried out 10 times vertically and 10 times horizontally using opposite sides of the sponges. For picker finger sampling, sponges were handled in the same manner as with carcass sampling and were used to swab an undelimited 200 cm2 area of the picker machines that included the rubber fingers. After sampling, all swabs were returned to their original bags. Thirty milliliters of scald water was collected using a sterile water “dippa” sampler with an integral handle (Bibby Sterilin, Staffordshire, UK). All swab and scald water samples were stored in a chilled container and transported to the laboratory within 2 h of sample collection.

Microbiological Analysis Forty milliliters of single strength BPW (Oxoid) was added to sponge samples and stomached for 90 s. For scald water samples, a 15-mL aliquot was added to 15 mL of double strength BPW (Oxoid). Sponge and scald water samples were incubated at 37°C for 18 to 24 h. Following incubation, aliquots of 0.5 and 0.1 mL of the BPW enriched samples were transferred to 10 mL of Tetrathionate (Difco, Sparks, MD) and 10 mL of Rappaport Vassiliadis broth (Difco) and incubated at 42°C for 18 to 24 h. After incubation, a loopful of Tetrathionate and Rappaport Vassiliadis broth were streaked onto XLT4

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nation (Ono and Yamamoto, 1999). Possible mechanisms of bacterial cross contamination during defeathering include aerosols (Allen et al., 2003a,b), direct contact between contaminated and uncontaminated carcasses (Mulder et al., 1978), and the action of the fingers of the picker machines (Clouser et al., 1995a,b; Berrang et al., 2001; Allen et al., 2003a,b). Studies of cross contamination during defeathering have focused on the change in prevalence that occurs before and after defeathering and the dissemination pattern of an indicator organism from an artificially contaminated bird to other birds during processing (Clouser et al., 1995a,b; Allen et al., 2003a,b). To our knowledge, no study has examined the relationship between Salmonella subtypes isolated before and after defeathering. Molecular typing is increasingly being used to complement conventional methodologies to elucidate bacterial transmission routes (De Cesare et al., 2001; Sander et al., 2001). Several studies have used molecular typing to understand the transmission of Salmonella within poultry production (De Cesare et al., 2001; Bailey et al., 2002; Liebana et al., 2002; Crespo et al., 2004). The application of such techniques will provide a better understanding of the mechanisms of cross contamination that may occur during defeathering. In this study, the breast feathers of turkeys were sampled before defeathering and the exposed breast skin of carcasses after defeathering to determine the extent of cross contamination occurring during defeathering. Serotyping and pulsed field gel electrophoresis (PFGE) were used to more specifically determine the relationship between Salmonella isolates obtained pre- and postdefeathering.

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Table 1. Prevalence of Salmonella-positive birds at pre- and postdefeathering Visit number 1 2 3 4 Total

Predefeathering no. positive birds/ no. sampled (%)

Postdefeathering no. positive birds/ no. sampled (%)

14/24 (58)a 14/50 (28)a 30/50 (60)a 24/50 (48)a 82/174 (47)a

24/24 (100)b 16/50 (32)a 25/50 (54)a 45/50 (90)b 110/174 (63)b

Statistical Analysis The McNemars test was performed using SAS 9 (SAS Institute Inc., 2004) to determine if there was a significant change in the prevalence of Salmonella-positive birds after defeathering for each visit. The McNemars test was also used to determine if there was a difference between the overall Salmonella prevalence before and after defeathering when all 4 visits were combined (Table 1).

a,b Percentages within a row lacking a common superscript differ (P < 0.05).

Pulsed Field Gel Electrophoresis of Salmonella Isolates Identified Salmonella isolates were subtyped by PFGE using the standardized protocol described by the Centers for Disease Control and Prevention National Molecular Subtyping network for Foodborne Disease Surveillance (CDC, 2001). Salmonella Branderup H9812 (ATCC#: BAA664) was used as the reference strain. The DNA macrorestriction fragments were resolved on 1% SeaKem Gold Agarose (Cambrex Bio Science Rockland Inc., Rockland, ME) in 0.5× Tris-borate EDTA. Then PFGE was carried out in 0.5× Tris-borate EDTA using the Chef Mapper PFGE system (BioRad, Hercules, CA) with recirculation at 14°C. Run time for electrophoresis was 18 h with initial switch time of 2.16 s and final switch time of 63.8 s. Following electrophoresis, gels were stained in ethidium bromide for 30 min. Destaining was carried out in reagent grade water for 60 min. Images of macrorestriction patterns on agarose gels were captured using an imager (Alpha Innotech UV imager, San Leandro, CA) and stored as .tif files. Macrorestriction patterns were compared using the BioNumerics Fingerprinting II Informatrix software (Version 3.0, Applied Maths, Austin, TX; BioRad). The similarity index of the isolates was calculated using the Dice correlation coefficient option of the software with a position tolerance of 1% and an optimization of 5%. The unweighted-pair group method using average linkages (UPGMA; Struelens, 1996) was used to construct dendrograms. A 70% similarity index was used to distinguish different clusters on the dendrograms.

Change in the Prevalence of SalmonellaPositive Birds at Pre- and Postdefeathering During visits 1 and 4, the Salmonella prevalence after defeathering was significantly higher (P < 0.05) than before defeathering (Table 1). There was no significant difference in Salmonella prevalence pre- and postdefeathering during visits 2 and 3. When all 4 visits were combined, there was an overall significant increase (P < 0.05) in Salmonella prevalence after defeathering.

PFGE Profiles Dendrogram analysis revealed 4 clusters each for visits 1, 2, and 3, as well as 5 clusters for visit 4 (results not shown). During all visits, serotyping correlated with clustering because only identical serotypes were in the same cluster. Using visit 3 as an example, Figure 1 shows the dendrogram generated. Clusters 1 and 2 contain only S. Hadar isolates, whereas clusters 3 and 4 contain S. Muenster isolates.

Relationship Among Salmonella Subtypes Isolated at Pre- and Postdefeathering, from Scald Water and the Rubber Fingers of the Picker Machines All Salmonella serotypes isolated after defeathering were also isolated before defeathering (Table 2). Salmonella serotypes with similar PFGE profiles were detected at pre- and postdefeathering and on the fingers of the picker machines during all 4 visits (Table 2). During visits 1 and 3, S. Hadar was isolated at the pre- and postdefeathering stages and on the fingers of the picker machines (Table 2). During visit 2, S. Schwarzengrund was isolated at pre- and postdefeathering and from the fingers of the picker machines (Table 2). During visit 4, S. Hadar was detected in scald water at pre- and postdefeathering and from the fingers of the picker machines (Table 2).

DISCUSSION The results of this study showed that there was transfer of Salmonella from live turkeys to carcass skin during defeathering. Cross contamination during defeathering

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(Difco) and Brilliant Green Sulfa (Difco) agar plates. Plates were incubated at 37°C for 24 to 48 h. Up to 6 presumptive Salmonella colonies, when available, were picked from the selective agar plates and streaked onto Lysine Iron Agar (Difco) and Triple Sugar Iron (Difco) agar slants and incubated at 37°C for 18 to 24 h. Suspect Salmonella colonies following biochemical screening were confirmed using the Sensititre method for automated identification of gram-negative organisms (AP 80, Trek Diagnostics, Cleveland, OH). Confirmed Salmonella isolates were serotyped by the National Veterinary Services Laboratories, Ames, IA.

RESULTS

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Figure 1. Dendrogram showing pulsed field gel electrophoresis (PFGE) profiles of Salmonella isolates from visit 2. Pre = Predefeathering; Post = postdefeathering; Fingers = fingers of picker machines.

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Table 2. Salmonella serotypes detected before and after defeathering, in scald water, and on the picker fingers during each visit1 Location

Serotypes

1

Pre

Bredeney Hadar Bredeney Hadar — Hadar Hadar Heidelberg Schwarzengrund Typhimurium Schwarzengrund Typhimurium — Schwarzengrund Hadar Muenster Hadar — Hadar Hadar Hadar Hadar Hadar

Post

2

Scald water Picker fingers Pre

Post

3

4

Scald water Picker fingers Pre Post Scald water Picker fingers Pre Post Scald water Picker fingers

PFGE profile A B A B B B E D C D C B F G F F F F F F

1 Pre = predefeathering; Post = postdefeathering; PFGE = pulsed field gel electrophoresis.

resulted in at least a similar number of birds being contaminated after defeathering as before (visits 2 and 3). On the other 2 occasions, there was a significant increase (P < 0.05) in the Salmonella prevalence after defeathering. This is in agreement with previously reported data from chicken and turkey carcass defeathering operations (Lillard, 1989, 1990; Clouser et al., 1995a,b) where Salmonella contamination after defeathering was observed to increase or remain the same. Characterizing isolates by serotyping and PFGE further facilitated the determination of the source and transmission route of Salmonella contamination during defeathering. This is in contrast to previous studies of cross contamination during defeathering where conclusions were limited by a lack of subtyping data. This is the first report of a clear link between Salmonella contamination of turkey feathers and subsequent carcass contamination. Previous studies have determined the Salmonella status of live birds by sampling feces (Rigby and Petit, 1980, 1981; Higgins et al., 1982). In another study, the increase in Campylobacter contamination of broilers that occurred after defeathering was attributed to the expulsion of feces from the cloacae by the peristaltic action of the fingers of the picker machines (Berrang et al., 2001). The results of the current study indicate that contaminated feathers should also be considered as a source of carcass contamination. Given that such contamination is on the external surface of bird, it is reasonable to suggest that it could be a more significant source of carcass contamination than feces that is normally internal. The detection of the same Salmonella subtype in scald water and at pre- and postdefeathering during visit 4 indicates that scald water is a vehicle for the transfer of

ACKNOWLEDGMENTS The authors gratefully acknowledge John Reber and Qiongzhen Li of North Dakota State University for statistical analysis and technical assistance during sample collection.

REFERENCES Allen, V. M., M. H. Hinton, D. B. Tinker, C. Gibson, G. C. Mead, and C. M. Wathes. 2003a. Microbial cross-contamination by airborne dispersion and contagion during defeathering of poultry. Br. Poult. Sci. 44:567–576.

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Visit

Salmonella between birds. This is supported by another study in which Escherichia coli K12 was used as an indicator organism to demonstrate cross contamination during scalding (Mead et al., 1994). Despite the difference in the scalding temperature between visits 1 and 2 (60°C) and visits 3 and 4 (63.5°C), it is unlikely that Salmonella was absent in scald water during visit 1 and 2. Salmonella has previously been isolated from scald water at 60°C (Nivas et al., 1973). Collecting 1 sample of scald water per visit during the current study may have been a limiting factor for Salmonella detection. Parameters such as sample volumes, sample numbers, and culturing procedures may also influence Salmonella detection in scald water (Cason et al., 2000). Further studies that incorporate these variables will provide more information on the potential of scald water as a vehicle for cross contamination during processing. The isolation of similar Salmonella subtypes from the fingers of the picker machines during all 4 visits (Table 2) and from birds at pre- and postdefeathering supports the role of the picker fingers in carcass cross contamination. This is in agreement with previous studies that have reported that the fingers of picker machines may facilitate bacterial cross contamination between carcasses (Clouser et al., 1995b; Berrang et al., 2001). Neither scald water nor the fingers of the picker machines are cleaned nor replaced between carcasses, supporting their potential for facilitating cross contamination between Salmonella-contaminated birds and Salmonellanegative birds when they are processed in succession. Scald water and the fingers of the picker machines may also contribute to the contamination of Salmonella-free flocks when they are processed following a Salmonellapositive flock. The results of this study show evidence for the possible transfer of Salmonella from turkey feathers to carcass skin during defeathering. This direct contaminating effect was greater during visits 1 and 4 when the Salmonella prevalence after defeathering increased significantly. Salmonella contamination on the turkeys feathers may, therefore, be a useful indicator of the potential for cross contamination during defeathering. Future strategies could focus on reducing the level of Salmonella on the feathers of live birds, thus minimizing the risk of cross contamination during defeathering.

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