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Efficacy of electrolyzed oxidizing water and lactic acid on the reduction of Campylobacter on naturally contaminated broiler carcasses during processing
PROCESSING, PRODUCTS, AND FOOD SAFETY Efficacy of electrolyzed oxidizing water and lactic acid on the reduction of Campylobacter on naturally contaminated broiler carcasses during processing G. Rasschaert,*1 V. Piessens,* P. Scheldeman,* S. Leleu,* A. Stals,* L. Herman,* M. Heyndrickx,*† and W. Messens*2 *Technology and Food Science Unit, Institute for Agricultural and Fisheries Research (ILVO), 9090 Melle, Belgium; and †Faculty of Veterinary Medicine, Department of Pathology, Bacteriology and Poultry Diseases, Ghent University, 9820 Merelbeke, Belgium ABSTRACT Campylobacter is the most commonly reported gastrointestinal bacterial pathogen in humans in many developed countries. During slaughter of broiler flocks, it is difficult to avoid contamination of broiler carcasses. This study aimed to quantify Campylobacter contamination on broiler carcasses at 5 points in the slaughter processing during the slaughter of a Campylobacter-colonized flock by real-time PCR and conventional enumeration. In addition, the decontamination effect of neutral electrolyzed oxidizing (EO) water and 1.5% lactic acid (pH 2.0) were evaluated. During processing, the Campylobacter counts on the carcasses declined toward the end of the processing line. The log counts on the carcasses as determined by quantitative real-time PCR (qPCR), decreased from 9.37 after scalding to 8.08 after the last cooling step. Enumeration of the campylobacters on plates revealed the same trend, although the counts per carcass were generally 3
logs lower. After scalding, a mean of 6.86 log cfu/carcass were counted, which decreased to 4.83 log cfu/carcass after the last cooling step. Submerging carcasses after scalding in EO water gave a significant reduction of 1.31 log cfu/carcass by enumeration on plates and a not significant reduction of 0.53 log cfu/carcass by qPCR. Treatment of the carcasses after the inside-outside bird washer led to reductions from 0.09 to 0.91 log cfu/carcass, although not significant. After submerging the carcasses in a 1.5% lactic acid solution, significant reductions of 1.62 and 1.24 log cfu/carcass by qPCR and enumeration, respectively, were observed. Spraying the carcasses with lactic acid led to nonsignificant reductions of 0.68 log cfu/carcass determined by qPCR and 0.26 log cfu/carcass by enumeration. Both EO water and lactic acid seem promising for implementation in poultry processing plants.
Key words: Campylobacter, broiler slaughterhouse, electrolyzed oxidizing water, lactic acid 2013 Poultry Science 92:1077–1084 http://dx.doi.org/10.3382/ps.2012-02771
INTRODUCTION In 2009, Campylobacter continued to be the most commonly reported gastrointestinal bacterial pathogen in humans in the European Union with 46 reported cases per 100,000 habitants (EFSA, European Centre for Disease Prevention and Control, 2011). Handling, preparation, and consumption of poultry are the main
©2013 Poultry Science Association Inc. Received September 13, 2012. Accepted December 24, 2012. 1 Corresponding author: [email protected] 2 Currently working as senior scientific officer with the Biological Hazards (BIOHAZ) Unit, European Food Safety Authority (EFSA), Parma, Italy. The views and findings in this article are solely those of the author and do not necessarily reflect the views or position of the European Food Safety Authority.
sources of human campylobacteriosis. Approximately 70% of all broiler flocks are colonized with Campylobacter (Herman et al., 2003; Rasschaert et al., 2006; EFSA, 2010). During slaughter, carcass contamination is difficult to avoid because the birds of colonized flocks may already be externally contaminated or can become contaminated via spilling the intestinal content directly or indirectly onto the carcasses (Newell et al., 2001; Slader et al., 2002; Herman et al., 2003; Rasschaert et al., 2006). To control Campylobacter throughout the poultry meat processing chain, intervention measures can be taken at the farm level, slaughterhouse level, or both. Many efforts have been made in the previous years to control Campylobacter during primary production, but these have either failed or have had only limited success (Hermans et al., 2011). Therefore, carcass decontamination during processing seems an easier method to
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reduce numbers of campylobacters on carcasses. It has been estimated that a 1 to 2 log reduction of the Campylobacter concentration on broiler carcasses would lead to a significant reduction in human campylobacteriosis cases (Rosenquistet al., 2003; Messenset al., 2007). During poultry processing, carcass decontamination may be achieved by physical or chemical treatment. Physical treatments, such as heat and low-temperature treatment, are effective in reducing the Campylobacter concentration on broiler carcasses, but have disadvantages regarding the appearance of the final product and considerable limitations for practical implementation at the plant. Irradiation is one of the most effective means to reduce Campylobacter contamination on carcasses and has a minimal effect on the sensory characteristics of the final poultry product, but European consumers’ acceptance of this practice is low [Corry and Atabay, 2001; EFSA, Panel on Biological Hazards (BIOHAZ), 2011]. Chemical decontamination also does not alter the final appearance of the meat, in most cases, and although applied in countries outside the European Union, chemical treatments are not authorized by the European Union [EFSA, Panel on Biological Hazards (BIOHAZ), 2011]. The EC Regulation No. 853/2004, however, allows decontamination treatments if the substance has been shown to be safe and effective. The EFSA was asked to evaluate the safety in use of lactic acid and sodium lactate for the decontamination of poultry carcasses, but was not able to formulate an opinion because of insufficient information submitted (EFSA, 2006a,b). Furthermore, scientific papers show inconsistent results on the use of lactic acid to reduce Campylobacter populations on artificially contaminated poultry meat (Zhao and Doyle, 2006; Lecompte et al., 2009; Riedel et al., 2009; Rajkovic et al., 2010). Another technique that is relatively easy to implement in a poultry slaughterhouse is decontamination of the carcasses by electrolyzed oxidizing (EO) water. The EO water is produced by the electrolysis of potable water supplemented with sodium chloride. A direct current with low voltage level leads to the formation of oxygen and chlorine radicals, which in turn activate reactive disinfectants (free chlorine). Depending on the applied technique, acidic and alkaline EO water, or neutral EO water, is formed. Acidic EO water is reported to reduce Campylobacter numbers on artificially contaminated carcasses (Park et al., 2002; Kim et al., 2005; Northcutt et al., 2007). Studies are lacking, however, on the use of lactic acid and EO water on naturally contaminated carcasses during poultry processing. The aim of this study was to evaluate the effect of neutral EO water and 1.5% L(+)lactic acid (pH 2.0) on decontamination of carcasses during processing of a Campylobacter-colonized broiler flock. To do so, the Campylobacter contamination on broiler carcasses was determined by quantitative methods at different processing steps with and without implementation of intervention measures.
MATERIALS AND METHODS Determination of the Campylobacter Status of the Flock One Campylobacter-positive flock (approximately 8,000 birds) was sampled during poultry processing. The status of the flock was determined 5 d before slaughter by collecting 30 cecal droppings in the poultry house. These cecal droppings were pooled together to 3 samples of 10 g each and were diluted 1:9 with buffered peptone water (CM509, Oxoid, Basingstoke, UK). After homogenization, 100 μL of each sample was plated in duplicate on modified charcoal cefoperazone deoxycholate agar (mCCDA, CM739 supplemented with SR155 and SR232, Oxoid) and incubated under microaerobic conditions at 41.5°C for 24 and 48 h. Typical colonies were counted to estimate the number of cfu in the cecum content.
Sampling in the Slaughterhouse The Belgian slaughterhouse was a poultry processing plant with a capacity of 10,000 birds per hour. First, the birds were hung manually, electrically stunned, then killed by exsanguination. In a second area, the carcasses were scalded in a counter-current flow scalding tank at a temperature of 52 ± 1°C before being mechanically plucked. In the evisceration room, the carcasses were eviscerated along with procedures to remove crop, neck, and internal organs. At the end of this processing line, the carcasses were washed inside and outside. The carcasses were air chilled at a temperature of approximately 3°C during 1 h 45 min. For the first 20 min, cold air was blown over the carcasses, after which the carcasses were chilled down for 85 min without further forced air flow. Approximately halfway through the process, water was sprayed on the carcasses to help cooling. Afterward there was an additional chilling step, during which the carcasses passed through a cooling tunnel (approximately −10°C) for 20 min with air streaming. During processing of the Campylobacter-colonized flock, whole carcasses were collected after scalding, before and after passing through the inside-outside bird washer (IOBW), after chilling, and again after the additional chilling step. At each of those processing points, 10 carcasses were taken randomly distributed over the processing of the entire flock. The efficacy of neutral EO water and lactic acid was manually evaluated during processing of this flock through submersion or spraying. The choice of substance and application manner was dependent of the place of implementation, which is further dealt with in the Discussion section. The EO water was evaluated after the scalding tank (submerging) and after the IOBW (either submerging or spraying). Lactic acid was only tested at the end of the processing line after the
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IOBW (either submerging or spraying). The EO water was generated using the Ecodis electrolysis technique (Ecodis, Schoten, Belgium) with a flow of 150 L∙h−1 with a neutral pH, and a (theoretical) residual chlorine level of 50 ppm. The residual chlorine level was measured during the tests (before and during treatment of each carcass). Ten carcasses were collected after scalding and were consecutively submerged for 3 min in a 15-L tank. During these 3 min, half of the volume of the tank was automatically replaced. An additional 20 carcasses, collected after the IOBW, were either submerged in EO water as described above or sprayed with EO water. Carcasses were sprayed for 3 min with a Gardena nozzle connected to the electrolysis cell with a flow of 150 L∙h−1. Lactic acid treatment consisted of submerging or spraying carcasses collected after the IOBW with 1.5% (wt/wt) L(+)-lactic acid pH 2.0 (FCC 80, PURAC Biochem, Gorinchem, the Netherlands). Ten carcasses were submerged for 3 min in a 15-L tank. After 5 carcasses, the content of the tank was manually refreshed. Another 10 carcasses were sprayed with 60 mL of lactic acid using an ordinary plant sprayer (bought in a supermarket). To assess the washing effect during submerging or spraying the carcasses with EO water or lactic acid, reference carcasses were used. After the scalding tank, where submersion of carcasses in EO water was evaluated, the reference carcasses were normally processed carcasses coming out of the scalding bath (as the washing effect can be considered maximal). After the IOBW, where both EO water and lactic acid were evaluated either by submersion or spraying, 20 carcasses were used for control, of which 10 were submerged in processing water and 10 were sprayed with processing water. This was performed the same way as described for the EO water. All carcasses were individually packed in sterile plastic bags. The carcasses were transported under cooled conditions and processed the same day (approximately 6 h between slaughter of the flock and processing in the laboratory).
Bacteriological Culture and Quantitative Real-Time PCR Because the carcasses collected after scalding were not eviscerated, the cloaca of these carcasses were plugged by a tampon to prevent intestinal leakage onto the carcasses during handling (Musgrove et al., 1997; Berrang et al., 2001). Each bag was drenched with 300 mL of buffered peptone water supplemented with 0.02% (vol/vol) Tween20 (Fukushima et al., 2007). The surface of each carcass was hand massaged for 1 min and shaken at 150 rpm for 1.5 min (VWR International, Leuven, Belgium). One milliliter of this rinse was plated onto a mCCDA plate. In addition, a dilution series was made from this suspension, and 100
100,
10−1,
10−3
and dilutions was plated in μL of the duplicate on mCCDA plates. After 24 and 48 h of incubation at 41.5°C under microaerobic conditions, colonies with a typical Campylobacter morphology on the mCCDA plates were counted by one person skilled for Campylobacter enumeration. Some colonies were picked and confirmed by a Campylobacter-specific PCR (Linton et al., 1996). The undiluted suspensions were also used to prepare crude cell lysates for quantitative realtime PCR (qPCR) as described by Botteldoorn et al. (2008). In the qPCR, the primers and probe of Lund et al. (2004) were used, and 1 µL of the crude cell lysate was added to the mix as described by Botteldoorn et al. (2008).
Statistical Analysis All statistical analyses were performed using Statistica (version 9.0; StatSoft, Tulsa, OK). The Tukey honestly significant difference test was used to determine significant differences between group means of different processing steps or intervention measures. The significance level α was set at 0.05.
RESULTS Campylobacter Counts on Carcasses During Processing of a CampylobacterColonized Flock The flock sampled in the slaughterhouse was excreting Campylobacter at approximately 9 log cfu∙g−1 ceca content at the farm. During slaughter of this Campylobacter-colonized flock, the Campylobacter contamination on the carcasses declined toward the end of the processing line (Figure 1). The counts on the carcasses, expressed as log cfu per carcass determined by qPCR, decreased from 9.37 after scalding to 8.50 before the IOBW to 8.08 after the IOBW to 8.00 after cooling. After the additional cooling step, the number of cfu increased to 8.52. Enumeration of the campylobacters on plates revealed the same trend, although the counts per carcass were generally 3 log lower (Figure 1). After scalding, 6.86 log cfu/carcass were counted, which decreased to 5.36 before the IOBW to 4.83 after the IOBW to 4.44 after cooling. Again, the number of campylobacters increased after the extra cooling step to 4.83. By both methods, the Campylobacter counts on the carcasses after the IOBW and after cooling were significantly lower than on the carcasses after scalding. The increase after the additional cooling step was not significant (Figure 1).
Campylobacter Counts on Carcasses After the Intervention Measures After scalding, submerging the carcasses in EO water gave a nonsignificant Campylobacter reduction of 0.53
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Figure 1. Campylobacter counts (in log cfu) per carcass during processing of a Campylobacter-colonized broiler flock, as determined by quantitative real-time PCR (full line) and enumeration on modified charcoal cefoperazone deoxycholate agar plates (dashed line). Vertical bars denote 0.95 CI. Common letters (A,B; a,b) on each curve indicate nonsignificant differences (P > 0.05). IOBW: inside-outside bird washer.
log cfu/carcass according to qPCR and a significant reduction of 1.31 log cfu/carcass when determined by plate counts (Figure 2). The mean residual chlorine during treatment of the 10 carcasses was 28.0 ppm with 14.3 ppm being the lowest value (Table 1). To assess the washing effect of the intervention measures implemented after the IOBW, carcasses submerged or sprayed with processing water were used as controls. The washing effect of both submerging or spraying was negligible as the differences were very small and nonsignificant (Figure 3). Submerging the carcasses in water led to a reduction of 0.04 log cfu/ carcass determined by qPCR and an increase of 0.13 log cfu/carcass determined by plate counts. Spraying the carcasses with water led to an increase of 0.51 and 0.22 log cfu/carcass, as determined by qPCR and enumeration on mCCDA plates, respectively. The use of EO water had no significant effect on the Campylobacter contamination of the carcasses (Figure 3). After submersion in EO water, an additional reduction of 0.09 log cfu/carcass (qPCR) and 0.35 log cfu/ carcass (plate counts) was observed compared with the control carcasses. Spraying the carcasses with EO water led to a reduction of 0.25 and 0.90 log cfu/carcass by qPCR and plate counts, respectively, compared with the controls. The mean residual chlorine during submerging of the 10 carcasses was 33.3 ppm, with values ranging from 0.88 to 46.6 ppm. The mean residual chlo-
rine during spraying of the 10 carcasses was 52.5 ppm, with only minor fluctuations (Table 1). Submersion in lactic acid was more efficient, as shown by a reduction of more than 1 log per carcass. Both
Figure 2. Campylobacter counts (in log cfu) per carcass after submerging the carcasses in electrolyzed oxidizing water (EO), determined by quantitative real-time PCR (full line) and enumeration on modified charcoal cefoperazone deoxycholate agar plates (dashed line). Vertical bars denote 0.95 CI. Common letters (A; a,b) on each curve indicate nonsignificant differences (P > 0.05).
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Time point Submerging in EO water: scalding tank Carcass 1 Carcass 2 Carcass 3 Carcass 4 Carcass 5 Carcass 6 Carcass 7 Carcass 8 Carcass 9 Carcass 10 Mean Submerging in EO water: IOBW2 Carcass 1 Carcass 2 Carcass 3 Carcass 4 Carcass 5 Carcass 6 Carcass 7 Carcass 8 Carcass 9 Carcass 10 Mean Spraying with EO water: IOBW 1 2 3 4 Mean 1NP
Before treatment carcass
During treatment carcass
After treatment carcass
39.9 32.6 14.3 20.5 36.4 34.2 30.5 29.9 27.6 NP1 29.5 30.7 37.3 38.4 44.1 38.9 39.4 31.1 33.7 39.6 34.1 36.7
37.8 21.5 21.9 19.6 28 31.2 28 26.4 21.7 NP 26.2 45.2 40.4 41.8 3.78 41.2 34.6 3.4 0.88 46.6 40.7 29.9 55.6 53.3 45.6 55.5 52.5
32.6 14.3 20.5 36.4 34.2 30.5 29.9 27.6 NP NP 28.3 37.3 38.4 44.1 38.9 39.4 31.1 33.7 39.6 34.1 41.4 37.8
= not performed because of technical problems. = inside-outside bird washer.
2IOBW
qPCR and enumeration on plates revealed a significant reduction of 1.62 and 1.24 log cfu/carcass, respectively, compared with the controls submerged in processing water. Spraying the carcasses with the lactic acid solution led to nonsignificant reductions of 0.68 log cfu/carcass as determined by qPCR and 0.26 log cfu/carcass as determined by plate counting.
DISCUSSION When processing Campylobacter-colonized flocks, carcass contamination is difficult to avoid. A solution is to decontaminate broiler carcasses during or after processing. This study evaluated the efficacy of neutral electrolyzed oxidizing water and lactic acid to decontaminate carcasses, originating from the same colonized flock. In addition to conventional enumeration on selective medium, qPCR was used to determine the number of campylobacters present on the carcasses. However, the aim of this study was not to compare the 2 enumeration techniques because this has already been accomplished and the published results demonstrated a high correlation (Hong et al., 2007; Rönner and Lindmark, 2007; Botteldoorn et al., 2008). To our knowledge, this is the
first study in which qPCR was used to quantify the number of campylobacters on broiler carcasses collected from several points within processing. Although the same downward trend in the number of campylobacters was observed with qPCR and plate counts, there was a difference of approximately 3 log in bacterial numbers between both methods at each step during processing. Real-time PCR tends to overestimate bacterial counts because the DNA of dead bacteria is also amplified, whereas the conventional enumeration underestimates the numbers because overgrowth of accompanying flora often hinders the counting of the campylobacters. Furthermore, viable but nonculturable campylobacters are only detected by qPCR. A difference of 1 to 3 log between both methods was also observed by Hong et al. (2007) and Botteldoorn et al. (2008). Differentiation between dead and live bacteria using qPCR may be achieved using propidium monoazide (Josefsen et al., 2010). A decreasing trend in the number of campylobacters on the carcasses was observed throughout the processing line by both enumeration techniques. This decline has also been observed in other studies (Oosterom et al., 1983; Izat et al., 1988; Berrang and Dickens, 2000), although in most studies a sharp decrease in the Cam-
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Figure 3. Campylobacter counts (in log cfu) per carcass after the inside-outside bird washer (IOBW) after various intervention measures determined by quantitative real-time PCR (in black) and enumeration on modified charcoal cefoperazone deoxycholate agar plates (in gray). Vertical bars denote 0.95 CI. Common letters (A,B; a,b) on each curve indicate nonsignificant differences (P > 0.05). LA = lactic acid; EO = electrolyzed oxidizing water.
pylobacter counts after scalding is reported due to the high temperatures of the scalding tank water. In those studies, the contamination of the carcasses after cooling was approximately the same as after scalding. However, in the present study, the Campylobacter counts on the carcasses after scalding were approximately 2 log higher than after cooling. Possible explanations are that in the studies of Oosterom et al. (1983) and Izat et al. (1988) the feathers of the postscald carcasses were manually removed and only the Campylobacter load on the skin was determined, whereas in the present study whole carcasses, including the feathers, were rinsed. This approach probably leads to a higher number of Campylobacter in the carcass rinses. Furthermore, Berrang and Dickens (2000) and Oosterom et al. (1983) mention a high-scald temperature (55°C to 58°C), compared with a low-scald temperature (52°C) in the slaughterhouse of the present study. In the present study, the total Campylobacter contamination of whole carcasses after the first cooling step was 4.44 log cfu, as determined by conventional enumeration on selective media. Although this contamination strongly depends on whether a Campylobacter-positive or -negative flock is examined, this Campylobacter load is in agreement with several other quantitative studies (Berrang and Dickens, 2000; Allen et al., 2007; Johannessen et al., 2007; Klein et al., 2007). This study shows that the first cooling step only has a limited,
nonsignificant effect. Air chilling has shown various results from no effect (Cudjoe and Kapperud, 1991) to nondetectable Campylobacter levels (Oosterom et al., 1983). This difference can be explained by the variable drying effect of air chilling, which causes physical stress for Campylobacter (Oosterom et al., 1983; Rosenquist et al., 2006; Klein et al., 2007). In the processing plant of our study, water was sprayed on the carcasses to enhance the cooling process and to avoid dehydration of the carcasses, which may explain the limited effect of cooling on the Campylobacter count. An additional cooling step of 20 min at −10°C with air streaming had an adverse though nonsignificant effect on the Campylobacter contamination of the carcasses, possibly due to a too short cooling time. The EO water was tested after the scalding tank and after the IOBW, as these are the obvious points to implement the use of EO water in a processing plant. After scalding, submersion of carcasses was evaluated, as the scalding water could theoretically be replaced by EO water. After the IOBW, both submerging and spraying of carcasses were evaluated, as the water used in the sprays of the washer could be replaced by EO water, or the carcasses could undergo an additional EO water washing step. Submerging the carcasses after scalding in EO water gave substantial, although not significant, reductions up to 1.31 log cfu/carcass by enumeration on mCCDA. Reductions from 0.09 to 0.90
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log cfu/carcass, all nonsignificant, were observed after treating the carcasses with EO water after the IOBW. In other studies, higher efficacies of EO water with artificially contaminated carcasses were described (Park et al., 2002). The current paper, however, addresses naturally contaminated carcasses, for which the bacteria in the crevices and feather follicles of the chicken skin may be more difficult to reach by the EO water. Furthermore, during this field trial, it became evident that is was difficult to maintain a constant value of residual chlorine, especially after the IOBW. This implies that the technology should be improved for large-scale applications. Intriguingly, reductions observed by conventional plate counting were consistently better than reductions measured by qPCR. This could be explained by the high oxidation potential of EO water damaging the bacterial cell membranes (Liao et al., 2007), but the DNA can probably still be detected in the qPCR. Lactic acid was tested only after the IOBW as it is only logical to chemically decontaminate the end product. Submerging carcasses in lactic acid led to significant reductions of 1.24 to 1.62 log cfu/carcass compared with the control carcasses submerged in water, whereas spraying only led to a nonsignificant reduction. The latter result is probably due to the use of an insufficient quantity of lactic acid during spraying. Other studies report varying results, from no reductions up to reductions of approximately 4 log (Lecompte et al., 2009; Riedel et al., 2009; Rajkovic et al., 2010). This may be attributed to differences between studies (e.g., concentrations of lactic acid, exposure times, artificial vs. naturally contaminated chicken parts, spraying vs. submerging, and enumeration immediately after treatment vs. enumeration 1 to 7 d after treatment). This last point is particularly important because the beneficial effect of applying lactic acid increases during chilled storage. In the present study, the effect of lactic acid was evaluated approximately 6 h after application. It would be interesting to evaluate the effect of lactic acid after application and rinsing off the chemical immediately or a few hours later [EFSA, Panel on Biological Hazards (BIOHAZ), 2011]. Remarkably, the qPCR showed better reductions for lactic-acid-treated carcasses than plate counting, for which we have no explanation. To conclude, the same decreasing trend in Campylobacter numbers on naturally contaminated chicken carcasses throughout the processing line was observed both by qPCR and conventional enumeration on mCCDA plates, although a 3-log difference between both methods was observed. The EO water seems to be a promising technique to reduce the number of Campylobacter on broiler carcasses during processing, although the technology needs improvement before large-scale application. Lactic acid also gave encouraging results, although a shorter time interval between application and rinsing may be considered and further evaluated. The efficacy of both EO water and lactic acid should be further evaluated on more flocks in more slaughterhous-
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es. During further optimization, the cost effectiveness should be determined for both intervention measures.
ACKNOWLEDGMENTS The authors thank the staff of the slaughterhouse and Ecodis for their kind cooperation. This study was funded by FOD R-04/002-CAMPY and IWT IWT050215. We thank Miriam Levenson (ILVO, Merelbeke, Belgium) for English language editing of this manuscript.
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bacter jejuni and Campylobacter coli in raw chicken meats using a real-time PCR method. J. Food Prot. 70:2015–2022. Izat, A. L., F. A. Gardner, J. H. Denton, and F. A. Golan. 1988. Incidence and level of Campylobacter jejuni in broiler processing. Poult. Sci. 67:1568–1572. Johannessen, G. S., G. Johnsen, M. Økland, K. S. Cudjoe, and M. Hofshagen. 2007. Enumeration of thermotolerant Campylobacter spp. from poultry carcasses at the end of the slaughterline. Lett. Appl. Microbiol. 44:92–97. Josefsen, M. H., C. Löfström, T. B. Hansen, L. S. Christensen, J. E. Olsen, and J. Hoorfar. 2010. Rapid quantification of viable Campylobacter bacteria on chicken carcasses using real-time PCR and propidium monoazide treatment as a tool for quantitative risk assessment. Appl. Environ. Microbiol. 76:5097–5104. Kim, C., Y.-C. Hung, and S. M. Russell. 2005. Efficacy of electrolyzed water in the prevention and removal of fecal material attachment and its microbicidal effectiveness during simulated industrial poultry processing. Poult. Sci. 84:1778–1784. Klein, G., F. Reich, L. Beckmann, and V. Atanassova. 2007. Quantification of thermophilic Campylobacter spp. in broilers during meat processing. Antonie van Leeuwenhoek 92:267–273. Lecompte, J.-Y., A. Collignan, S. Sarter, E. Cardinale, and A. Kondjoyan. 2009. Decontamination of chicken surfaces inoculated with Listeria innocua, Salmonella enteritidis and Campylobacter jejuni by contact with a concentrated lactic acid solution. Br. Poult. Sci. 50:307–317. Liao, L. B., W. M. Chen, and X. M. Xiao. 2007. The generation and inactivation mechanism of oxidation–reduction potential of electrolyzed oxidizing water. J. Food Eng. 78:1326–1332. Linton, D., A. J. Lawson, R. J. Owen, and J. Stanley. 1996. Rapid identification by PCR of the genus Campylobacter and of five Campylobacter species enteropathogenic for man and animals. Res. Microbiol. 147:707–718. Lund, M., S. Nordentoft, L. Pedersen, and M. Madsen. 2004. Detection of Campylobacter spp. in chicken fecal samples by real-time PCR. J. Clin. Microbiol. 42:5125–5132. Messens, W., E. Hartnett, X. Gellynck, J. Viaene, D. Halet, L. Herman, and K. Grijspeerdt. 2007. Quantitative risk assessment of human campylobacteriosis through the consumption of chicken meat in Belgium. XVIII European Symposium on the Quality of Poultry Meat and XII European Symposium on the Quality of Eggs and Egg Products, September 2–5, Prague, Czech Republic. Musgrove, M. T., J. A. Cason, D. L. Fletcher, N. J. Stern, N. A. Cox, and J. S. Bailey. 1997. Effect of cloacal plugging on microbial recovery from partially processed broilers. Poult. Sci. 76:530–533.
Newell, D. G., J. E. Shreeve, M. Toszeghy, S. Domingue, S. Bull, T. Humphrey, and G. Mead. 2001. Changes in the carriage of Campylobater strains by poultry carcasses during processing in abattoirs. Appl. Environ. Microbiol. 67:2636–2640. Northcutt, J., D. Smith, K. D. Ingram, A. Hinton Jr., and M. Musgrove. 2007. Recovery of bacteria from broiler carcasses after spray washing with acidified electrolyzed water or sodium hypochlorite solutions. Poult. Sci. 86:2239–2244. Oosterom, J., S. Notermans, H. Karman, and G. B. Engels. 1983. Origin and prevalence of Campylobacter jejuni in poultry processing. J. Food Prot. 46:339–344. Park, H., Y. C. Hung, and R. E. Brackett. 2002. Antimicrobial effect of electrolyzed water for inactivating Campylobacter jejuni during poultry washing. Int. J. Food Microbiol. 72:77–83. Rajkovic, A., N. Tomic, N. Smigic, M. Uyttendaele, P. Ragaert, and F. Devliegere. 2010. Survival of Campylobacter jejuni on raw chicken legs packed in high-oxygen or high carbon dioxide atmosphere after the decontamination with lactic acid/sodium lactate buffer. Int. J. Food Microbiol. 140:201–206. Rasschaert, G., K. Houf, J. Van Hende, and L. De Zutter. 2006. Campylobacter contamination during poultry slaughter in Belgium. J. Food Prot. 69:27–33. Riedel, C. T., L. Brøndsted, H. Rosenquist, S. N. Haxgart, and B. B. Christensen. 2009. Chemical decontamination of Campylobacter jejuni on chicken skin and meat. J. Food Prot. 72:1173–1180. Rönner, A. C., and H. Lindmark. 2007. Quantitative detection of Campylobacter jejuni on fresh chicken carcasses by real-time PCR. J. Food Prot. 70:1373–1378. Rosenquist, H., N. L. Nielsen, H. M. Sommer, B. Nørrung, and B. B. Christensen. 2003. Quantitative risk assessment of human campylobacteriosis associated with thermophilic Campylobacter species in chickens. Int. J. Food Microbiol. 83:87–103. Rosenquist, H., H. M. Sommer, N. L. Nielsen, and B. B. Christensen. 2006. The effect of slaughter operations on the contamination of chicken carcasses with thermotolerant Campylobacter. Int. J. Food Microbiol. 108:226–232. Slader, J., G. Domingue, F. Jørgensen, K. Mcalpine, R. J. Owen, F. J. Bolton, and T. J. Humphrey. 2002. Impact of transport crate reuse and of caching and processing Campylobacter and Salmonella contamination of broiler chickens. Appl. Environ. Microbiol. 68:713–719. Zhao, T., and M. P. Doyle. 2006. Reduction of Campylobacter jejuni on chicken wings by chemical treatments. J. Food Prot. 69:762–767.
logs lower. After scalding, a mean of 6.86 log cfu/carcass were counted, which decreased to 4.83 log cfu/carcass after the last cooling step. Submerging carcasses after scalding in EO water gave a significant reduction of 1.31 log cfu/carcass by enumeration on plates and a not significant reduction of 0.53 log cfu/carcass by qPCR. Treatment of the carcasses after the inside-outside bird washer led to reductions from 0.09 to 0.91 log cfu/carcass, although not significant. After submerging the carcasses in a 1.5% lactic acid solution, significant reductions of 1.62 and 1.24 log cfu/carcass by qPCR and enumeration, respectively, were observed. Spraying the carcasses with lactic acid led to nonsignificant reductions of 0.68 log cfu/carcass determined by qPCR and 0.26 log cfu/carcass by enumeration. Both EO water and lactic acid seem promising for implementation in poultry processing plants.
Key words: Campylobacter, broiler slaughterhouse, electrolyzed oxidizing water, lactic acid 2013 Poultry Science 92:1077–1084 http://dx.doi.org/10.3382/ps.2012-02771
INTRODUCTION In 2009, Campylobacter continued to be the most commonly reported gastrointestinal bacterial pathogen in humans in the European Union with 46 reported cases per 100,000 habitants (EFSA, European Centre for Disease Prevention and Control, 2011). Handling, preparation, and consumption of poultry are the main
©2013 Poultry Science Association Inc. Received September 13, 2012. Accepted December 24, 2012. 1 Corresponding author: [email protected] 2 Currently working as senior scientific officer with the Biological Hazards (BIOHAZ) Unit, European Food Safety Authority (EFSA), Parma, Italy. The views and findings in this article are solely those of the author and do not necessarily reflect the views or position of the European Food Safety Authority.
sources of human campylobacteriosis. Approximately 70% of all broiler flocks are colonized with Campylobacter (Herman et al., 2003; Rasschaert et al., 2006; EFSA, 2010). During slaughter, carcass contamination is difficult to avoid because the birds of colonized flocks may already be externally contaminated or can become contaminated via spilling the intestinal content directly or indirectly onto the carcasses (Newell et al., 2001; Slader et al., 2002; Herman et al., 2003; Rasschaert et al., 2006). To control Campylobacter throughout the poultry meat processing chain, intervention measures can be taken at the farm level, slaughterhouse level, or both. Many efforts have been made in the previous years to control Campylobacter during primary production, but these have either failed or have had only limited success (Hermans et al., 2011). Therefore, carcass decontamination during processing seems an easier method to
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reduce numbers of campylobacters on carcasses. It has been estimated that a 1 to 2 log reduction of the Campylobacter concentration on broiler carcasses would lead to a significant reduction in human campylobacteriosis cases (Rosenquistet al., 2003; Messenset al., 2007). During poultry processing, carcass decontamination may be achieved by physical or chemical treatment. Physical treatments, such as heat and low-temperature treatment, are effective in reducing the Campylobacter concentration on broiler carcasses, but have disadvantages regarding the appearance of the final product and considerable limitations for practical implementation at the plant. Irradiation is one of the most effective means to reduce Campylobacter contamination on carcasses and has a minimal effect on the sensory characteristics of the final poultry product, but European consumers’ acceptance of this practice is low [Corry and Atabay, 2001; EFSA, Panel on Biological Hazards (BIOHAZ), 2011]. Chemical decontamination also does not alter the final appearance of the meat, in most cases, and although applied in countries outside the European Union, chemical treatments are not authorized by the European Union [EFSA, Panel on Biological Hazards (BIOHAZ), 2011]. The EC Regulation No. 853/2004, however, allows decontamination treatments if the substance has been shown to be safe and effective. The EFSA was asked to evaluate the safety in use of lactic acid and sodium lactate for the decontamination of poultry carcasses, but was not able to formulate an opinion because of insufficient information submitted (EFSA, 2006a,b). Furthermore, scientific papers show inconsistent results on the use of lactic acid to reduce Campylobacter populations on artificially contaminated poultry meat (Zhao and Doyle, 2006; Lecompte et al., 2009; Riedel et al., 2009; Rajkovic et al., 2010). Another technique that is relatively easy to implement in a poultry slaughterhouse is decontamination of the carcasses by electrolyzed oxidizing (EO) water. The EO water is produced by the electrolysis of potable water supplemented with sodium chloride. A direct current with low voltage level leads to the formation of oxygen and chlorine radicals, which in turn activate reactive disinfectants (free chlorine). Depending on the applied technique, acidic and alkaline EO water, or neutral EO water, is formed. Acidic EO water is reported to reduce Campylobacter numbers on artificially contaminated carcasses (Park et al., 2002; Kim et al., 2005; Northcutt et al., 2007). Studies are lacking, however, on the use of lactic acid and EO water on naturally contaminated carcasses during poultry processing. The aim of this study was to evaluate the effect of neutral EO water and 1.5% L(+)lactic acid (pH 2.0) on decontamination of carcasses during processing of a Campylobacter-colonized broiler flock. To do so, the Campylobacter contamination on broiler carcasses was determined by quantitative methods at different processing steps with and without implementation of intervention measures.
MATERIALS AND METHODS Determination of the Campylobacter Status of the Flock One Campylobacter-positive flock (approximately 8,000 birds) was sampled during poultry processing. The status of the flock was determined 5 d before slaughter by collecting 30 cecal droppings in the poultry house. These cecal droppings were pooled together to 3 samples of 10 g each and were diluted 1:9 with buffered peptone water (CM509, Oxoid, Basingstoke, UK). After homogenization, 100 μL of each sample was plated in duplicate on modified charcoal cefoperazone deoxycholate agar (mCCDA, CM739 supplemented with SR155 and SR232, Oxoid) and incubated under microaerobic conditions at 41.5°C for 24 and 48 h. Typical colonies were counted to estimate the number of cfu in the cecum content.
Sampling in the Slaughterhouse The Belgian slaughterhouse was a poultry processing plant with a capacity of 10,000 birds per hour. First, the birds were hung manually, electrically stunned, then killed by exsanguination. In a second area, the carcasses were scalded in a counter-current flow scalding tank at a temperature of 52 ± 1°C before being mechanically plucked. In the evisceration room, the carcasses were eviscerated along with procedures to remove crop, neck, and internal organs. At the end of this processing line, the carcasses were washed inside and outside. The carcasses were air chilled at a temperature of approximately 3°C during 1 h 45 min. For the first 20 min, cold air was blown over the carcasses, after which the carcasses were chilled down for 85 min without further forced air flow. Approximately halfway through the process, water was sprayed on the carcasses to help cooling. Afterward there was an additional chilling step, during which the carcasses passed through a cooling tunnel (approximately −10°C) for 20 min with air streaming. During processing of the Campylobacter-colonized flock, whole carcasses were collected after scalding, before and after passing through the inside-outside bird washer (IOBW), after chilling, and again after the additional chilling step. At each of those processing points, 10 carcasses were taken randomly distributed over the processing of the entire flock. The efficacy of neutral EO water and lactic acid was manually evaluated during processing of this flock through submersion or spraying. The choice of substance and application manner was dependent of the place of implementation, which is further dealt with in the Discussion section. The EO water was evaluated after the scalding tank (submerging) and after the IOBW (either submerging or spraying). Lactic acid was only tested at the end of the processing line after the
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IOBW (either submerging or spraying). The EO water was generated using the Ecodis electrolysis technique (Ecodis, Schoten, Belgium) with a flow of 150 L∙h−1 with a neutral pH, and a (theoretical) residual chlorine level of 50 ppm. The residual chlorine level was measured during the tests (before and during treatment of each carcass). Ten carcasses were collected after scalding and were consecutively submerged for 3 min in a 15-L tank. During these 3 min, half of the volume of the tank was automatically replaced. An additional 20 carcasses, collected after the IOBW, were either submerged in EO water as described above or sprayed with EO water. Carcasses were sprayed for 3 min with a Gardena nozzle connected to the electrolysis cell with a flow of 150 L∙h−1. Lactic acid treatment consisted of submerging or spraying carcasses collected after the IOBW with 1.5% (wt/wt) L(+)-lactic acid pH 2.0 (FCC 80, PURAC Biochem, Gorinchem, the Netherlands). Ten carcasses were submerged for 3 min in a 15-L tank. After 5 carcasses, the content of the tank was manually refreshed. Another 10 carcasses were sprayed with 60 mL of lactic acid using an ordinary plant sprayer (bought in a supermarket). To assess the washing effect during submerging or spraying the carcasses with EO water or lactic acid, reference carcasses were used. After the scalding tank, where submersion of carcasses in EO water was evaluated, the reference carcasses were normally processed carcasses coming out of the scalding bath (as the washing effect can be considered maximal). After the IOBW, where both EO water and lactic acid were evaluated either by submersion or spraying, 20 carcasses were used for control, of which 10 were submerged in processing water and 10 were sprayed with processing water. This was performed the same way as described for the EO water. All carcasses were individually packed in sterile plastic bags. The carcasses were transported under cooled conditions and processed the same day (approximately 6 h between slaughter of the flock and processing in the laboratory).
Bacteriological Culture and Quantitative Real-Time PCR Because the carcasses collected after scalding were not eviscerated, the cloaca of these carcasses were plugged by a tampon to prevent intestinal leakage onto the carcasses during handling (Musgrove et al., 1997; Berrang et al., 2001). Each bag was drenched with 300 mL of buffered peptone water supplemented with 0.02% (vol/vol) Tween20 (Fukushima et al., 2007). The surface of each carcass was hand massaged for 1 min and shaken at 150 rpm for 1.5 min (VWR International, Leuven, Belgium). One milliliter of this rinse was plated onto a mCCDA plate. In addition, a dilution series was made from this suspension, and 100
100,
10−1,
10−3
and dilutions was plated in μL of the duplicate on mCCDA plates. After 24 and 48 h of incubation at 41.5°C under microaerobic conditions, colonies with a typical Campylobacter morphology on the mCCDA plates were counted by one person skilled for Campylobacter enumeration. Some colonies were picked and confirmed by a Campylobacter-specific PCR (Linton et al., 1996). The undiluted suspensions were also used to prepare crude cell lysates for quantitative realtime PCR (qPCR) as described by Botteldoorn et al. (2008). In the qPCR, the primers and probe of Lund et al. (2004) were used, and 1 µL of the crude cell lysate was added to the mix as described by Botteldoorn et al. (2008).
Statistical Analysis All statistical analyses were performed using Statistica (version 9.0; StatSoft, Tulsa, OK). The Tukey honestly significant difference test was used to determine significant differences between group means of different processing steps or intervention measures. The significance level α was set at 0.05.
RESULTS Campylobacter Counts on Carcasses During Processing of a CampylobacterColonized Flock The flock sampled in the slaughterhouse was excreting Campylobacter at approximately 9 log cfu∙g−1 ceca content at the farm. During slaughter of this Campylobacter-colonized flock, the Campylobacter contamination on the carcasses declined toward the end of the processing line (Figure 1). The counts on the carcasses, expressed as log cfu per carcass determined by qPCR, decreased from 9.37 after scalding to 8.50 before the IOBW to 8.08 after the IOBW to 8.00 after cooling. After the additional cooling step, the number of cfu increased to 8.52. Enumeration of the campylobacters on plates revealed the same trend, although the counts per carcass were generally 3 log lower (Figure 1). After scalding, 6.86 log cfu/carcass were counted, which decreased to 5.36 before the IOBW to 4.83 after the IOBW to 4.44 after cooling. Again, the number of campylobacters increased after the extra cooling step to 4.83. By both methods, the Campylobacter counts on the carcasses after the IOBW and after cooling were significantly lower than on the carcasses after scalding. The increase after the additional cooling step was not significant (Figure 1).
Campylobacter Counts on Carcasses After the Intervention Measures After scalding, submerging the carcasses in EO water gave a nonsignificant Campylobacter reduction of 0.53
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Figure 1. Campylobacter counts (in log cfu) per carcass during processing of a Campylobacter-colonized broiler flock, as determined by quantitative real-time PCR (full line) and enumeration on modified charcoal cefoperazone deoxycholate agar plates (dashed line). Vertical bars denote 0.95 CI. Common letters (A,B; a,b) on each curve indicate nonsignificant differences (P > 0.05). IOBW: inside-outside bird washer.
log cfu/carcass according to qPCR and a significant reduction of 1.31 log cfu/carcass when determined by plate counts (Figure 2). The mean residual chlorine during treatment of the 10 carcasses was 28.0 ppm with 14.3 ppm being the lowest value (Table 1). To assess the washing effect of the intervention measures implemented after the IOBW, carcasses submerged or sprayed with processing water were used as controls. The washing effect of both submerging or spraying was negligible as the differences were very small and nonsignificant (Figure 3). Submerging the carcasses in water led to a reduction of 0.04 log cfu/ carcass determined by qPCR and an increase of 0.13 log cfu/carcass determined by plate counts. Spraying the carcasses with water led to an increase of 0.51 and 0.22 log cfu/carcass, as determined by qPCR and enumeration on mCCDA plates, respectively. The use of EO water had no significant effect on the Campylobacter contamination of the carcasses (Figure 3). After submersion in EO water, an additional reduction of 0.09 log cfu/carcass (qPCR) and 0.35 log cfu/ carcass (plate counts) was observed compared with the control carcasses. Spraying the carcasses with EO water led to a reduction of 0.25 and 0.90 log cfu/carcass by qPCR and plate counts, respectively, compared with the controls. The mean residual chlorine during submerging of the 10 carcasses was 33.3 ppm, with values ranging from 0.88 to 46.6 ppm. The mean residual chlo-
rine during spraying of the 10 carcasses was 52.5 ppm, with only minor fluctuations (Table 1). Submersion in lactic acid was more efficient, as shown by a reduction of more than 1 log per carcass. Both
Figure 2. Campylobacter counts (in log cfu) per carcass after submerging the carcasses in electrolyzed oxidizing water (EO), determined by quantitative real-time PCR (full line) and enumeration on modified charcoal cefoperazone deoxycholate agar plates (dashed line). Vertical bars denote 0.95 CI. Common letters (A; a,b) on each curve indicate nonsignificant differences (P > 0.05).
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Time point Submerging in EO water: scalding tank Carcass 1 Carcass 2 Carcass 3 Carcass 4 Carcass 5 Carcass 6 Carcass 7 Carcass 8 Carcass 9 Carcass 10 Mean Submerging in EO water: IOBW2 Carcass 1 Carcass 2 Carcass 3 Carcass 4 Carcass 5 Carcass 6 Carcass 7 Carcass 8 Carcass 9 Carcass 10 Mean Spraying with EO water: IOBW 1 2 3 4 Mean 1NP
Before treatment carcass
During treatment carcass
After treatment carcass
39.9 32.6 14.3 20.5 36.4 34.2 30.5 29.9 27.6 NP1 29.5 30.7 37.3 38.4 44.1 38.9 39.4 31.1 33.7 39.6 34.1 36.7
37.8 21.5 21.9 19.6 28 31.2 28 26.4 21.7 NP 26.2 45.2 40.4 41.8 3.78 41.2 34.6 3.4 0.88 46.6 40.7 29.9 55.6 53.3 45.6 55.5 52.5
32.6 14.3 20.5 36.4 34.2 30.5 29.9 27.6 NP NP 28.3 37.3 38.4 44.1 38.9 39.4 31.1 33.7 39.6 34.1 41.4 37.8
= not performed because of technical problems. = inside-outside bird washer.
2IOBW
qPCR and enumeration on plates revealed a significant reduction of 1.62 and 1.24 log cfu/carcass, respectively, compared with the controls submerged in processing water. Spraying the carcasses with the lactic acid solution led to nonsignificant reductions of 0.68 log cfu/carcass as determined by qPCR and 0.26 log cfu/carcass as determined by plate counting.
DISCUSSION When processing Campylobacter-colonized flocks, carcass contamination is difficult to avoid. A solution is to decontaminate broiler carcasses during or after processing. This study evaluated the efficacy of neutral electrolyzed oxidizing water and lactic acid to decontaminate carcasses, originating from the same colonized flock. In addition to conventional enumeration on selective medium, qPCR was used to determine the number of campylobacters present on the carcasses. However, the aim of this study was not to compare the 2 enumeration techniques because this has already been accomplished and the published results demonstrated a high correlation (Hong et al., 2007; Rönner and Lindmark, 2007; Botteldoorn et al., 2008). To our knowledge, this is the
first study in which qPCR was used to quantify the number of campylobacters on broiler carcasses collected from several points within processing. Although the same downward trend in the number of campylobacters was observed with qPCR and plate counts, there was a difference of approximately 3 log in bacterial numbers between both methods at each step during processing. Real-time PCR tends to overestimate bacterial counts because the DNA of dead bacteria is also amplified, whereas the conventional enumeration underestimates the numbers because overgrowth of accompanying flora often hinders the counting of the campylobacters. Furthermore, viable but nonculturable campylobacters are only detected by qPCR. A difference of 1 to 3 log between both methods was also observed by Hong et al. (2007) and Botteldoorn et al. (2008). Differentiation between dead and live bacteria using qPCR may be achieved using propidium monoazide (Josefsen et al., 2010). A decreasing trend in the number of campylobacters on the carcasses was observed throughout the processing line by both enumeration techniques. This decline has also been observed in other studies (Oosterom et al., 1983; Izat et al., 1988; Berrang and Dickens, 2000), although in most studies a sharp decrease in the Cam-
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Figure 3. Campylobacter counts (in log cfu) per carcass after the inside-outside bird washer (IOBW) after various intervention measures determined by quantitative real-time PCR (in black) and enumeration on modified charcoal cefoperazone deoxycholate agar plates (in gray). Vertical bars denote 0.95 CI. Common letters (A,B; a,b) on each curve indicate nonsignificant differences (P > 0.05). LA = lactic acid; EO = electrolyzed oxidizing water.
pylobacter counts after scalding is reported due to the high temperatures of the scalding tank water. In those studies, the contamination of the carcasses after cooling was approximately the same as after scalding. However, in the present study, the Campylobacter counts on the carcasses after scalding were approximately 2 log higher than after cooling. Possible explanations are that in the studies of Oosterom et al. (1983) and Izat et al. (1988) the feathers of the postscald carcasses were manually removed and only the Campylobacter load on the skin was determined, whereas in the present study whole carcasses, including the feathers, were rinsed. This approach probably leads to a higher number of Campylobacter in the carcass rinses. Furthermore, Berrang and Dickens (2000) and Oosterom et al. (1983) mention a high-scald temperature (55°C to 58°C), compared with a low-scald temperature (52°C) in the slaughterhouse of the present study. In the present study, the total Campylobacter contamination of whole carcasses after the first cooling step was 4.44 log cfu, as determined by conventional enumeration on selective media. Although this contamination strongly depends on whether a Campylobacter-positive or -negative flock is examined, this Campylobacter load is in agreement with several other quantitative studies (Berrang and Dickens, 2000; Allen et al., 2007; Johannessen et al., 2007; Klein et al., 2007). This study shows that the first cooling step only has a limited,
nonsignificant effect. Air chilling has shown various results from no effect (Cudjoe and Kapperud, 1991) to nondetectable Campylobacter levels (Oosterom et al., 1983). This difference can be explained by the variable drying effect of air chilling, which causes physical stress for Campylobacter (Oosterom et al., 1983; Rosenquist et al., 2006; Klein et al., 2007). In the processing plant of our study, water was sprayed on the carcasses to enhance the cooling process and to avoid dehydration of the carcasses, which may explain the limited effect of cooling on the Campylobacter count. An additional cooling step of 20 min at −10°C with air streaming had an adverse though nonsignificant effect on the Campylobacter contamination of the carcasses, possibly due to a too short cooling time. The EO water was tested after the scalding tank and after the IOBW, as these are the obvious points to implement the use of EO water in a processing plant. After scalding, submersion of carcasses was evaluated, as the scalding water could theoretically be replaced by EO water. After the IOBW, both submerging and spraying of carcasses were evaluated, as the water used in the sprays of the washer could be replaced by EO water, or the carcasses could undergo an additional EO water washing step. Submerging the carcasses after scalding in EO water gave substantial, although not significant, reductions up to 1.31 log cfu/carcass by enumeration on mCCDA. Reductions from 0.09 to 0.90
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log cfu/carcass, all nonsignificant, were observed after treating the carcasses with EO water after the IOBW. In other studies, higher efficacies of EO water with artificially contaminated carcasses were described (Park et al., 2002). The current paper, however, addresses naturally contaminated carcasses, for which the bacteria in the crevices and feather follicles of the chicken skin may be more difficult to reach by the EO water. Furthermore, during this field trial, it became evident that is was difficult to maintain a constant value of residual chlorine, especially after the IOBW. This implies that the technology should be improved for large-scale applications. Intriguingly, reductions observed by conventional plate counting were consistently better than reductions measured by qPCR. This could be explained by the high oxidation potential of EO water damaging the bacterial cell membranes (Liao et al., 2007), but the DNA can probably still be detected in the qPCR. Lactic acid was tested only after the IOBW as it is only logical to chemically decontaminate the end product. Submerging carcasses in lactic acid led to significant reductions of 1.24 to 1.62 log cfu/carcass compared with the control carcasses submerged in water, whereas spraying only led to a nonsignificant reduction. The latter result is probably due to the use of an insufficient quantity of lactic acid during spraying. Other studies report varying results, from no reductions up to reductions of approximately 4 log (Lecompte et al., 2009; Riedel et al., 2009; Rajkovic et al., 2010). This may be attributed to differences between studies (e.g., concentrations of lactic acid, exposure times, artificial vs. naturally contaminated chicken parts, spraying vs. submerging, and enumeration immediately after treatment vs. enumeration 1 to 7 d after treatment). This last point is particularly important because the beneficial effect of applying lactic acid increases during chilled storage. In the present study, the effect of lactic acid was evaluated approximately 6 h after application. It would be interesting to evaluate the effect of lactic acid after application and rinsing off the chemical immediately or a few hours later [EFSA, Panel on Biological Hazards (BIOHAZ), 2011]. Remarkably, the qPCR showed better reductions for lactic-acid-treated carcasses than plate counting, for which we have no explanation. To conclude, the same decreasing trend in Campylobacter numbers on naturally contaminated chicken carcasses throughout the processing line was observed both by qPCR and conventional enumeration on mCCDA plates, although a 3-log difference between both methods was observed. The EO water seems to be a promising technique to reduce the number of Campylobacter on broiler carcasses during processing, although the technology needs improvement before large-scale application. Lactic acid also gave encouraging results, although a shorter time interval between application and rinsing may be considered and further evaluated. The efficacy of both EO water and lactic acid should be further evaluated on more flocks in more slaughterhous-
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es. During further optimization, the cost effectiveness should be determined for both intervention measures.
ACKNOWLEDGMENTS The authors thank the staff of the slaughterhouse and Ecodis for their kind cooperation. This study was funded by FOD R-04/002-CAMPY and IWT IWT050215. We thank Miriam Levenson (ILVO, Merelbeke, Belgium) for English language editing of this manuscript.
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