D.J. CALDWELL1
J.A. BYRD2
S.M. STEVENS1
D.J. NISBET2
A.P. McELROY3
1Departments of Poultry Science and Veterinary Pathobiology, Texas A&M University, U.S.A.
2USDA-ARS-SPARC, College Station, Texas, U.S.A.
3Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, U.S.A.
The safety of our food supply continues to be a matter of paramount importance for regulatory agencies, researchers, health care professionals, and consumers world-wide. Human food borne illness is the cause of between 6.9- 34.9 billion dollars in economic losses each year in the U.S. alone (Buzby and Roberts, 1997). These losses were calculated based on an estimated 76 million annual cases of food borne illness which resulted in 325,000 hospitalizations and 5,000 deaths (Mead et al., 1999). Using these estimates for incidence, approximately one-fourth of all U.S. citizens will contract a food borne disease during the next year (Tauxe, 2002). Salmonella and Campylobacter are the two predominant human food borne pathogens associated with poultry and poultry products. Together they account for 90% of the entire reported bacterial food borne illnesses worldwide (Thorns, 2000). In 2003, Campylobacter (5273 cases) accounted for over 33% of all confirmed foodborne-related diseases (USDA-FSIS, 2005). Campylobacter is responsible for more than 2 million incidences of enteritis per year within the United States (Smith, 2002). With the implementation of the USDA-FSIS mandated pathogen reduction program known as HACCP (Hazard Analysis Critical Control Point program) in 1996, commercial poultry producers and processors were, for the first time, required to comply with performance standards for achieving reductions of Salmonella on processed broiler carcasses. While turkey processing facilities are currently only held to a ground turkey standard as opposed to the whole carcass standard for broilers, a whole carcass standard for turkeys is likely forthcoming.
While some of the responsibility for controlling both Salmonella and Campylobacter on processed poultry and poultry products falls within the realm of grow-out or live production, the processing plant has borne the majority of responsibility when evaluated on a historical basis. While some areas in commercial poultry processing exist as potential sites for the dissemination or cross-contamination of microorganisms, bird washers and immersion chill tanks, when managed properly, can be sites of pathogen reduction. The immersion chiller environment has also been identified as a critical point for bacterial reductions on poultry carcasses (James et al., 1992; White et al., 1997). The immersion chilling systems of processing facilities consist of large, open, ice cold, common water baths that operate under counter current flow and constant agitation. These common baths are required to effectively drop the carcass temperature to 4°C in 8 hours for birds weighing over 8 pounds (FSIS, 1999). Antimicrobial agents are routinely applied to the chill water to assist with the killing of bacteria. Chlorine is the most common antimicrobial compound utilized in turkey and broiler plants within the U.S.
Mead co-workers (1975) demonstrated a ten-fold reduction of faecal and spoilage bacteria on post-processed carcasses when a 20 ppm solution of chlorine was used in the plant. The factors that affect the successful attachment of a bacterium to carcass tissues include the pH, water flow, temperature, and organic material present. These factors also greatly affect the efficacy of chlorine in these and other applications. Similarly, other microbicides, such as ozone (Kim et al., 1999) and ClO2 (Baran et al., 1973) have been shown to be effective in the reduction of bacteria on carcasses during chilling. While the effectiveness of gas (Cl2) and hypochlorite (OCl-) forms of chlorination are highly dependent on the pH of chiller water, the efficacy of ClO2 is not. Whatever the chosen microbicide used in the chiller, proper management of the environment is critical for achieving reductions on finished carcasses.
These and other studies, almost exclusively conducted within broiler plants, have demonstrated that if managed properly, the immersion chiller can be a means to create an inhibitory environment for microorganisms, effectively reducing bacterial load on the finished carcasses. Many have assumed that these findings and observations in broiler processing plants can be directly extrapolated or extended to turkey plants. For this reason, little attention has been given to microbial intervention strategies within commercial turkey processing. Due to the major differences between the processing environments in broiler and turkey processing plants and the major differences in the live production or rearing environments between turkeys and broilers, we contend this argument is flawed.
A major research focus of our laboratories over the past several years has been to identify practical pre- and post-harvest microbial interventions for commercial poultry producers and processors. To this end, we have worked closely with the commercial turkey industry within the U.S. since 2000 to accomplish this objective. The present manuscript reports findings from our work with three commercial turkey processing facilities in the U.S. aimed at attempting to identify best management practices and effective management-based immersion chiller microbial intervention strategies. Sampling within these facilities was conducted initially in 2002 and followed up with additional sampling in 2004. These facilities were located in geographically distinct regions of the U.S. and all sampling was completed during the spring of each year of sampling.
Materials and methods
Overview
In this investigation we performed a microbial survey of commercial turkey processing facilities with the objective of evaluating the effectiveness of intervention strategies for reducing Salmonella on commercially processed turkeys. Sites of evaluation in this survey included prior to and following immersion chilling. To determine the effectiveness of the selected interventions currently being used within these three turkey processing facilities, carcasses were rinsed for bacterial recovery at pre- and post-immersion chill sampling sites.
Immersion chiller design and intervention strategies
All plants contained similar immersion chillers designed by the same manufacturer (Morris and Associates, Raleigh, North Carolina). These chillers were designed for counter current flow and the number of individual chillers varied by each plant’s line configuration. Each facility utilized a different approach for microbial intervention within the immersion chilling system.
Plants 1 and 2 chose chlorine dioxide (ClO2) as a principle chiller microbicide. Plant 2 also utilized an additional intervention for its chilling system that consisted of filtration and ozone application to re-circulated water. Plant 3 chose conventional chlorine application for microbial control within its chilling system by first applying gas chlorine (Cl2) and then adding supplemental sodium hypochlorite (NaOCl) to meet the established plant target range for measurable chlorine within chiller water during processing.
Carcass rinse sample collection
Sample collection and culture of the recovered rinse fluid for the specific isolation of Salmonella throughout this study was based on the published USDA-FSIS proposed “Mega-Reg” guidelines for sampling and culture as described in the Federal Register (Federal Register, 1996), with slight modification. Sample collection and recovery of Campylobacter followed accepted protocols as described below. On each day of sampling within each facility (two sampling days in 2002 and one sampling day in 2004), approximately 100 carcass rinse samples were obtained from both pre- and post-immersion chiller sampling sites for determination of Salmonella and Campylobacter incidence from recovered rinse fluid. Carcasses were removed from the processing line or immediately upon exit from the immersion chiller in an alternating fashion between the pre- and post-sampling sites. Carcasses were removed with an individual pair of sterilized latex gloves and placed into a sterilized polypropylene bag for carcass rinsing. To rinse the individual carcasses, 200 ml of sterile buffered peptone water (BPW; pH 7.2) was added to each individual bag, and the carcasses were rinsed using inverted rotation 30 times each. Approximately one hundred ml of the carcass rinse fluid was then aseptically recovered by allowing the rinse fluid to accumulate in a corner of the rinse bag. The corner and a pair of scissors were sprayed with 70% alcohol, wiped down, and the corner was then cut. The rinse fluid was then allowed to drain into a sterilized polypropylene collection bottle. All sample collection bottles were placed on wet ice and transported back to our laboratories for the initiation of bacteriologic culture, which began approximately 24 h following the time of sample collection. The samples were treated equally and recovery incidence between sampling locations was compared. Transport-related effects on sensitivity were not a critical concern in the present investigation. To ascertain the effectiveness of the chosen microbial interventions in each facility for achieving reductions in bacterial incidence on processed carcasses, certain parameters were also measured directly in chiller water. The effectiveness of the management approach in each facility was determined by measuring chill water pH, total chlorine, free chlorine, or chlorine dioxide levels, where applicable, at the entrance and exit of chillers during sampling. These measurements are presented with microbial recovery data below.
Salmonella Culture
For the specific culture of Salmonella, 30 ml of the recovered rinse fluid was combined and pre-enriched in an additional 30 ml of BPW for 24 hours at 37°C. Following pre-enrichment, 100 µl of each sample was sterilely transferred to a sterile culture tube containing 10 ml of Rappaport-Vassiliadis (RV) enrichment broth and incubated for 24 hours at 42°C. Following enrichment, each sample was then streaked onto modified lysine iron agar (MLIA) plating medium for the specific recovery of Salmonella. Plates were incubated for 24 hours at 37°C. Suspect colonies were confirmed biochemically (triple sugar iron and lysine iron slants) and serologically (Salmonella o antisera (poly o; a-i).
Campylobacter Culture
For selective culture of Campylobacter from carcass rinse fluid and chill water samples, 10 mL of collected rinse fluid from each individual sample was enriched in Bolton’s complete enrichment medium for 24 hours at 42°C. Following selective enrichment, each sample was streaked onto Campy-Cefex agar plating medium and all plates were incubated for 48 hours at 42°C, as described by Stern and co-workers (Stern et al., 1992). All suspect Campylobacter isolates were confirmed as belonging to the genus Campylobacter by examination of cellular morphology and motility on wet mount under phase contrast microscopy. The colonies that met cellular morphology and motility requirements were further characterized by a positive reaction on latex agglutination test kit. All Campylobacter culture procedures were conducted under a modified atmosphere containing 5% O2, 10% CO2, and 85% N2.
Statistical Analysis
Differences in bacterial incidence (+/-) for Salmonella or Campylobacter recovered by carcass rinsing at specific sampling sites were compared using the Chi-square test of independence and significant (P<0.05) differences are reported.
Results
Plant 1
Measurement of pH within Plant 1 during all sampling days in 2002 and 2004 revealed pH values that consistently remained above pH 7. These observations likely had no impact on microbial intervention within the chill tank due to the use of chlorine dioxide (ClO2; Table 1). During 2002 sampling, ClO2 levels were measured consistently between 0.5 and 1.5 ppm throughout both days of sampling. Despite a slightly lower ClO2 level of 0.2 ppm during the first sampling set of 2004, the recorded measurement of 2.32 ppm in the second sampling set was the highest measured for all sampling sets within this facility (Table 1). Bacterial recovery incidence for Plant 1 revealed reductions in Salmonella post-chill carcass incidence when comparing between pre- and post-chill sites of sampling during both 2002 and 2004 sampling (Table 1). During day 1 of sampling in 2002, when pre- and post-chill Salmonella incidence data were compared, significant differences in post-chill incidence were observed during both the first (P<.05) and second sampling periods (P<.05). Despite an overall lower pre-chill Salmonella burden during day 2 of sampling in 2002, significant reductions (P<.05) were observed during both the first and second sampling times. Similarly, subsequent sampling of carcasses within this facility during 2004 revealed a consistent trend of reduced Salmonella incidence (P<.05) following immersion chilling in this facility (Table 1). Recovery incidence in Plant 1 also revealed reductions in Campylobacter post-chill carcass incidence when comparing between pre- and post-chill sites in 5 of six sampling sets (Table 2). Despite an overall lower pre-chill Campylobacter burden during day 2 of sampling in 2002, significant reductions (P<.05) were observed during both the sampling dates.
Plant 2
During all measurements obtained during both 2002 and 2004 sampling, chlorine dioxide levels (0.04-0.3 ppm) in the third chill tank (only site of chlorine dioxide application within the immersion chilling system) were lower than established plant target levels (1-2 ppm; Table 3). These levels were also lower than the levels measured in Plant 1, which utilized similar equipment and management for this microbial intervention strategy. Despite being comparatively lower and below target, levels measured on each day of sampling were consistent and apparently effective in reducing Salmonella on post-chill processed turkey carcasses (Table 3). Measurements of pH were consistently within the 8.3-8.5 range during all days of sampling (Table 3). Similar to Plant 1 however, due to the choice of ClO2 as a primary chill tank intervention strategy, pH levels had no impact on the observed data. The lower pH and ClO2 levels were still associated with reduced Campylobacter on post-chill processed turkey carcasses when the concentrations were greater than 0.14 ppm for ClO2 (Table 4). 
Concentrations of ClO2 of 0.1 ppm or lower did not significantly reduce the recovery of Campylobacter and even had a significant increase with chill water containing 0.1 ppm ClO2 of the 2004 sampling dates. However, it should be noted that 4 of the 6 sampling sets had lower initial Campylobacter ¬positive pre-chill carcasses than any of those observed in Plant 1. Bacterial load likely influenced our observations in this regard.
As mentioned above, this facility also filtered and applied ozone to re-circulated chiller water to remove solids and further reduce bacteria in re-used chiller water. This combined approach likely led to the minimal amount of visible organic matter and subsequent significant reductions of Salmonella incidence on post-chill carcasses throughout both days of sampling in 2002 in this plant. Comparison between pre- and post-chill Salmonella incidence levels during two sequential days of commercial turkey processing during 2002 and a single day of processing during 2004 within Plant 2 are reported in Table 3. On each of two days of sampling in this particular processing facility during 2002, significant reductions (P<.05) in Salmonella recovery were observed following immersion chilling as compared to pre-chill sampling at each sampling time period (Table 3). A lower overall pre-chill burden of Salmonella on carcasses coupled with consistently low levels of ClO2 in chiller water were likely contributory to dissimilar findings during sampling in 2004, as pre-chill and post-chill isolation frequencies, while uniformly low, did not differ (P>0.05).
Plant 3
Overall, observations of chill tank measurements within Plant 3 revealed total chlorine levels to be below the established plant target range (25-40 ppm) but generally consistent (range: 9.8-17.8 ppm) during each day of sampling in 2002 and 2004 (Table 5). Despite these generally lower total chlorine levels and the relatively high pH (average measurements between 7.9 and 8.5) of chill tank water during both days of sampling, free chlorine levels were consistently elevated. These levels were actually much higher than recorded values in other plants we’ve sampled, which have had more favourable total chlorine and pH profiles. Visual inspection of chill water throughout sampling in 2002 and 2004 revealed low to very low levels of organic matter and solids. Such observations were likely effective for maintaining higher free chlorine levels in chiller water despite more alkaline pH conditions (Table 5).
Salmonella pre- and post-chill incidence levels recorded for Plant 3 revealed significant reductions (P<.05) in post-chill incidence when compared to pre-chill incidence during each sampling period on both days of sampling in 2002 (Table 5). Similarly, during the first sampling set of 2004, chill water parameters were associated with a significant reduction (P<.05) of Salmonella on carcasses at the post-chill sampling site. A drop in measurable free chlorine (from 6.86 to 0.13 ppm) was associated with near-equivalent pre- and post-chill isolation frequencies during the second sampling set of 2004 sampling (Table 5). Campylobacter pre- and post-chill incidence levels recorded for Plant 3 measured significant reductions (P<.001) in post-chill incidence during 3 of 6 total sampling sets when compared to pre-chill incidence (Table 6). Again, it should be noted that 5 of the 6 sampling sets had lower initial Campylobacter-positive pre-chill carcasses when compared to Plant 1 or other facilities we’ve sampled.
Discussion
Taken together, data collected during this investigation indicate that properly managed immersion chilling systems consistently remediate Salmonella and Campylobacter from processed carcasses during commercial turkey processing. During the present investigation, two distinct forms of chlorine were compared as primary chill tank microbial intervention strategies: chlorine gas (Cl2; with supplemental sodium hypochlorite (NaOCl)) or chlorine dioxide (ClO2). Both forms of chlorinating chiller water were determined to be effective based upon our collected observations. When considering traditional forms of chlorine administration, free available chlorine levels are generally measured at lower levels than that of total chlorine. Data from the present investigation support previous findings which report the effectiveness of free chlorine as a bactericidal agent being dependent upon the conditions under which it is used in the processing environment (Mead and Thomas, 1973). Such conditions include the concentration of total chlorine applied, contact time with the carcass, temperature, pH, and the presence or absence of organic matter or solids. Control of chill water pH is critical to improving the effectiveness of conventional forms of chlorine used within commercial immersion chilling systems. However, in this investigation Plants 1 and 2 utilized ClO2 as a chiller microbial intervention strategy, which is unaffected by pH conditions that render conventional forms of chlorine ineffective as microbicides. Some commercial turkey processing plants we’ve worked with have very effectively used carbon dioxide (CO2) to acidify re-circulated chiller water as a means of pH control. Plant 3 in this investigation did not utilize this or a similar method of controlling pH of chiller water, and not surprisingly, pH levels of immersion chiller water at all times measured were consistently above 7 (7.9-8.4). Nonetheless, this facility was observed to maintain generally high free chlorine levels in chiller water, especially early in the day during processing. This observation was likely attributable to maintenance of low to very low levels of organic matter and solids in chill water throughout any given processing day. This association of low organic solids and elevated free chlorine was also supported by Mead and Thomas (1973).
Within all three facilities participating in this investigation, properly managed immersion chilling systems were observed to be a very effective microbial reduction strategy for achieving reductions in Salmonella and Campylobacter on processed carcasses. These findings are similar to other investigations conducted in broiler processing facilities where the immersion chilling environment has been identified as a critical focus for controlling bacterial contamination on poultry carcasses (James et al., 1992; White et al., 1997).
Measured values of applied microbicides within immersion chiller water in our investigation were clearly associated with the effectiveness of bacterial remediation on processed turkey carcasses, similar to the findings of Mead and co-workers (1975) in their investigations during broiler processing. Importantly, assessment of chill tank management within each plant was predictive of recorded microbiologic data. Obtained data indicated that facilities implementing the most stringent immersion chilling management practices were associated with the most effective Salmonella and Campylobacter reductions on processed carcasses.
Further, if assessments of management reflected poor practices or oversight, microbiologic data were similarly reflective of such trends. Data from this investigation suggest that properly managed immersion chilling systems with appropriate microbial intervention strategies are effective in reducing the incidence of Salmonella and Campylobacter on post-chill processed turkey carcasses.
References are available on request
From Proceedings of the “Midwest Poultry Federation Convention”, St. Paul, Minnesota, U.S.A.
