- Email: [email protected]
The effect of sampling time on the levels of micro-organisms recovered from broiler carcasses in a commercial slaughter plant
ARTICLE IN PRESS FOOD MICROBIOLOGY Food Microbiology 21 (2004) 59–65
www.elsevier.nl/locate/jnlabr/yfmic
The effect of sampling time on the levels of micro-organisms recovered from broiler carcasses in a commercial slaughter plant P. Whytea,*, K. McGilla, C. Monahanb, J.D. Collinsa a
Veterinary Public Health and Food Safety Laboratory, Department of Large Animal Clinical Studies, Faculty of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland b Dawn Farm Foods Ltd., Naas Co., Kildare, Ireland Received 14 November 2002; accepted 10 March 2003
Abstract Changes in the levels of indicator micro-organisms and Listeria on broiler carcasses were determined at various stages of commercial broiler slaughter and processing. In addition, the effect of time of day that sampling was carried out within the plant on the microbial profile of samples was evaluated. Counts of total aerobic organisms (37 C and 22 C), Enterobacteriaceae, E. coli and Staphylococcus aureus on broiler skin samples were consistently lower on samples obtained during evening visits to the co-operating broiler slaughter plant when compared with corresponding morning samples. Counts of S. aureus were significantly lower for both morning and evening samples taken following final carcass rinsing when compared to pre-evisceration equivalents (Po0:05). The number of total viable aerobic bacteria, Enterobacteriaceae and E. coli in scald tank water samples taken early each morning prior to processing start-up were significantly lower than corresponding samples obtained after flocks had been processed where an average of 3912 and 42 475 birds had been processed on morning and evening visits, respectively (Po0:05). The prevalence of Listeria on broiler skin samples consistently increased for both morning and evening samples through the various stages of processing. For example, the prevalence on samples taken in the morning following defeathering, final carcass rinsing, in-line chilling and final carcass chilling was 10%, 40%, 56% and 72%, respectively. The most frequently recovered species of Listeria from these samples were L. innocua (86%), L. monocytogenes (6%), L. grayi (6%) and L. seeligeri (2%). r 2003 Elsevier Ltd. All rights reserved. Keywords: Poultry microbiology; Indicator organisms; Enterobacteriaceae; Escherichia coli; Listeria; Staphylococcus
1. Introduction The handling and consumption of poultry meat is recognized as a major cause of foodborne illness in humans globally (Anonymous, 2000; Fitzgerald et al., 2001), particularly when eaten raw, undercooked or recontaminated and stored following cooking (Bryan, 1988; White et al., 1996). Processed broiler carcasses and raw poultry products are frequently contaminated with pathogenic micro-organisms of public health significance including, Salmonella (Panisello et al., 2000), thermophilic campylobacters (Jacobs-Reitsma, 1997, 2000; Anonymous, 2002), and Listeria (Capita et al., 2001; Uyttendaele et al., 1999). *Corresponding author. Tel.: +353-1-7166074; fax: +353-17166090. E-mail address: [email protected] (P. Whyte). 0740-0020/03/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0740-0020(03)00040-6
The microbiological status of processed poultry carcasses has been reported to be dependent on several key factors, namely: the level of contamination from live birds, numbers and genera of pathogenic or indicator organisms introduced at pre-harvest phases, and the extent of occurrence of contamination and cross-contamination during processing (Abu-Ruwaida et al., 1994). The control of contamination during slaughter and processing has been identified as critical in relation to the prevalence of pathogenic micro-organisms on end products (Mead et al., 1993). Clouser et al. (1995a) identified transport crates, scald water, picker fingers, evisceration equipment, chill water and personnel as potential sources of cross-contamination during slaughter. Cross-contamination can also occur during rearing, transport and holding together with the principal processing stages (Clouser et al., 1995b).
ARTICLE IN PRESS 60
P. Whyte et al. / Food Microbiology 21 (2004) 59–65
Upton (1996) noted that the extent to which contamination occurred on carcasses and the composition of the resulting flora reflected the hygiene standards at processing level, together with the initial microbiological status of the animal. Smoot and Pierson (1997) regarded the application of microbiological criteria using indicator organisms as useful for foods such as poultry which were repeatedly implicated in foodborne disease outbreaks, as the presence of certain organisms indicated the possibility of occurrence of microbiological hazards. However, the limitations associated with the use of indicator organisms in poultry and meat processing as a means of assessment of food safety have been previously documented by Goepfert (1976). The potential microbiological hazards in meat products cannot be assessed nor demonstrated reliably with the use of acceptable levels based on total counts or indicator organisms (Brown and Baird-Parker, 1982). However, the analysis of foods of animal origin for the presence or levels of generic Escherichia coli has been widely accepted as an indicator of faecal contamination, while failure to detect this organism cannot guarantee the absence of other enteric pathogens (Silliker and Gabis, 1976; Brown and Baird-Parker, 1982). The levels of Enterobacteriaceae on poultry carcasses have also been routinely used as a means to indicate inadequate or unhygienic processing or inappropriate handling or storage conditions which may result in the proliferation of a wide range of pathogenic and toxinproducing bacteria, including Salmonella (Brown and Baird-Parker, 1982; Roberts et al., 1995). Total aerobic counts have been recommended as a useful tool in the microbiological assessment of food safety, as high mesophilic counts (25–37 C) may indicate unsatisfactory sanitation or the use of heavily contaminated raw materials (Thatcher and Clark, 1968). Lillard et al. (1984) and Al-Mohizea et al. (1994) also acknowledged the benefit of aerobic plate counts as a criterion in assessing the hygiene of poultry processing plants. The effect of processing techniques on rates of crosscontamination by pathogenic organisms, including, Salmonella and Campylobacter, has been demonstrated by increased prevalences reported following carcass slaughter and dressing procedures (Waldroup et al., 1992; Jones et al., 1991). The mechanisms by which contamination occurs during processing, other than immersion in scald and chill waters, have been described by Schuler and Badenhop (1972) and Carson et al. (1987), who demonstrated carcass–carcass and equipment–carcass contacts to be significant routes in the spread of pathogenic organisms. The present study was carried out to determine the levels of a range of indicator micro-organisms on broiler carcasses sampled at various stages of a commercial
slaughter process. In addition, the study examined whether the microbiological load present on slaughtered broiler carcasses was dependent on the time of day that samples were obtained in the plant. It is frequently assumed that the microbiological profile of broiler carcasses disimproves over time in a processing plant as a result of the build up of high numbers of organisms on equipment surfaces and their dissemination within the plant on to carcasses. However, few studies have been carried out to ascertain and quantify the effect that time of sampling may have on the profile of carcasses processed on the same day within a slaughter plant.
2. Methods 2.1. Description and sampling procedures of the broiler processing plant The slaughter plant used in the current study was part of an integrated broiler operation and processed on average 60 000 birds per day. Processing commenced at 7.30 AM each morning and continued for 12 h or 1.5 shifts. The average throughput ranged from 5000 to 5500 birds per hour. Birds were supplied for slaughter by contract growers to the plant at ages which varied between 35 and 54 days depending on carcass weight requirements. In total, 15 visits to the slaughter plant were made over a 4-month period. Sampling was carried out on the first load of birds of each days throughput for the first 10 visits with samples from the remaining five visits taken from loads processed 7–8 h after daily production had commenced. During each visit, five neck skin samples were aseptically collected from different carcasses at four separate sampling points within the plant. Samples were collected each day from carcasses within the same flock at each sampling point. The sampling points used for the current study were: (i) immediately following defeathering at the transfer point to the evisceration line, (ii) after evisceration and final carcass washing, (iii) following in-line primary air chilling and (iv) after exiting the blast freezing tunnel (final carcass chilling). The equipment used in the plant was specifically designed for commercial broiler processing (Stork, N.V., Naarden, The Netherlands). Carcass temperatures entering the in-line primary air chill were slightly lower than those observed in live birds and averaged 39 C. Carcasses were air chilled in this system for approximately 45–60 min with temperatures of exiting carcasses ranging from +2 C to +8 C depending on mean carcass weights. The temperature of carcasses entering the freezing tunnel averaged +4 C while those exiting the chill dropped to Ca. 2 C. In addition, 100 ml scald water samples were taken each morning from the tank at the point where birds exited each morning prior to the
ARTICLE IN PRESS P. Whyte et al. / Food Microbiology 21 (2004) 59–65
commencement of production and again after the load sampled had been slaughtered. The scald tank used was a single stage unit operating at a temperature of 52 C. All samples were transported to the laboratory at p+4 C and microbiological analysis was carried out on the day of sampling. 2.2. Microbiological analysis For neck skins, 25 g of each sample was aseptically weighed and placed in sterile stomacher bags to which 225 ml of 0.1% peptone water was added. The samples were homogenized for 1 min each using a stomacher (Lab Blender 400 series, Seward Medical, UK). Serial 10-fold dilutions were then prepared by transferring 1 ml of this solution to 9 ml tubes of 0.1% peptone water. Once samples had been stomached and 10-fold dilutions prepared, the levels of total aerobic mesophilic (37 C) and psychrophilic (22 C) organisms, Enterobacteriaceae, E. coli and Staphylococcus aureus and the prevalence of Listeria spp. was established. For total aerobic and Enterobacteriaceae counts, 1 ml of the relevant dilutions were placed in duplicate plates and approximately 20 ml of molten standard plate count agar (Oxoid, Basingstoke, UK) or violet red bile glucose agar (Oxoid, Basingstoke, UK) were added after which the dilutions and molten agar were mixed and allowed to solidify. Total count plates were incubated for 48 and 72 h at 37 C and 22 C, respectively, before colonies were counted. Violet-red bile glucose agar plates were incubated at 37 C for 18–24 h after which characteristic purple haloed colonies were counted. E. coli were enumerated from stomached samples using a method previously described (Anonymous, 1994). Briefly, 0.5 ml of each dilution was spread in duplicate on surface mounted membrane filters (Gelman Sciences, MN, USA) which were placed on the surface of tryptone bile agar plates (Oxoid, Basinstoke, UK). The plates were then incubated overnight at 44 C. After incubation, the membrane filters were removed from the agar plates and placed on equivalent sized filter papers (Whatman International, Maidstone, UK) that had been soaked in 2 ml volumes of indole detection reagent (5% pdimethylaminobenzaldehyde dissolved in 1 N HCl) (Sigma-Aldrich, Dorset, UK). Indole positive colonies developed a pink-purple coloration on the membrane filter surfaces within 20 min and were presumptively counted as E. coli. and S. aureus were presumptively enumerated on the basis of their appearance on BairdParker medium (Oxoid, Basingstoke, UK). Briefly, 0.1 ml volumes of relevant dilutions were spread on duplicate plates which were then incubated for 48 h at 37 C. A sample of 10–15 colonies from each sampling day’s plates were confirmed as coagulase-positive S. aureus using a latex agglutination test (Murex Diagnostics, Kent, UK). Listeria spp. were isolated from
61
samples by adding 50 ml of the stomached sample to an equal volume of double strength pre-enrichment broth (Oxoid, Basingstoke, UK) and incubating for 18–24 h at 30 C. The samples were then selectively enriched by inoculating 0.1 ml volumes of the pre-enrichment media into broths containing a Listeria selective supplement (Oxoid, Basinstoke, UK). Samples were subcultured onto Oxford formulation Listeria selective plates (Oxoid, Basingstoke, UK) which were incubated at 37 C for 24 and 48 h. The confirmation and identification of suspect Listeria colonies on the solid media was then carried out by transferring to tryptone soya agar (Oxoid, Basingstoke, UK) and incubating for 24 h at 37 C. Using Henrys’ illumination technique of holding the plates at a 45 angle to a light source, typical Listeria colonies exhibited a blue to blue-grey colour. Speciation was carried out using the API biochemical profiling system (Biomerieux, Lyon, France). Scald tank water samples were also analysed for total viable counts, Enterobacteriaceae and E. coli levels by making up 10-fold serial dilutions and plating out onto the various selective and differential media as described above. 2.3. Statistical analysis All microbial counts obtained from neck skin samples were transformed to log10 values for subsequent data analysis. Unpaired Student’s t-tests were used to compare counts from both neck skin and scald tank water samples. Significance was defined at the 95% level (Pp0:05). The ‘‘StatView 5’’ programme (SAS Institute Inc., USA) was used for all statistical analyses.
3. Results The levels of all of the indicator organisms enumerated from neck skins were consistently lower on samples obtained during evening visits than those from morning samplings (Table 1). Significantly lower counts of total mesophilic and psychrophilic organisms were observed in evening samples at the final carcass wash, in-line chill exit and final chill exit sampling points when compared with corresponding morning samples (Po0:05). The mean number of Enterobacteriaceae was higher but not statistically significant, on evening samples taken following in-line chilling than in equivalent samples taken during morning visits with 3.79 and 3.37 log10 cfu/g, respectively. Similarly, significant differences in the levels of E. coli and S. aureus were observed between morning and evening samples taken immediately following final carcass washing (Po0:05). Interestingly, levels of Enterobacteriaceae, E. coli and S. aureus found on either sample sets did not statistically differ to finished product sampled exiting the final air chill.
ARTICLE IN PRESS P. Whyte et al. / Food Microbiology 21 (2004) 59–65
62
Table 1 Summary of results of mean total viable mesophilic and psychrophilic aerobes, Enterobacteriaceae, Escherichia coli and Staphylococcus aureus counts from neck skin samples taken during 10 morning and 5 evening visits to the broiler processing plant Sampling time
Total viable counts
SP 1 After defeathering SP 2 After final carcase washing SP 3 In-line chill exit SP 4 Final chill exit
Enterobacteriaceae
E. coli
S. aureus
37 C AM
37 C PM
22 C AM
22 C PM
AM
PM
AM
PM
AM
PM
5.27 (0.27) 4.87a (0.30) 5.12a (0.44) 5.08a (0.26)
5.01* (0.36) 4.44b** (0.61) 4.47b** (0.26) 4.58b** (0.26)
5.05 (0.24) 4.79a (0.32) 5.12a (0.43) 5.15a (0.29)
4.88* (0.41) 4.38b** (0.60) 4.46b** (0.37) 4.64b*,** (0.09)
3.28* (0.39) 3.37* (0.44) 3.79** (0.30) 3.50*,** (0.39)
3.25 (0.28) 3.17 (0.19) 3.37 (0.13) 3.33 (0.15)
3.01 (0.56) 3.26a (0.43) 3.35 (0.48) 3.28 (0.63)
2.90 (0.21) 2.95b (0.35) 3.11 (0.23) 3.20 (0.31)
3.04* (0.56) 2.48a** (0.43) 2.48** (0.48) 2.30** (0.63)
2.89* (0.47) 2.09b** (0.21) 2.40** (0.30) 2.35** (0.28)
n ¼ 50 for morning sampling points (AM), n ¼ 25 for evening sampling points (PM). Counts expressed as log10 cfu/g of mean values. ( )=standard deviation. *Denotes significance in counts between sampling points for each organism category taken during morning visits (Po0.05). **Denotes significance in counts between sampling points for each organism category taken during evening visits (Po0.05). a Denotes significance of morning counts for each organism category taken at the same sampling point (Po0.05). b Denotes significance of afternoon counts for each organism category taken at the same sampling point (Po0.05).
When counts on neck skins obtained during evening visits were examined, it was noted that significant reductions were observed between the defeathering and carcass washing stages for total mesophilic and psychrophilic counts (Po0:05), This trend was also observed between these sampling points in the plant for S. aureus counts on both morning and evening samples. The recovery of Listeria from neck skin samples obtained both during the morning and evening consistently increased through the various stages of processing, with ascending prevalences observed between sampling points 1, 2, 3 and 4, respectively (Table 2). The number of positive samples recovered during morning visits increased from 5% to 72% for sampling points 1 and 4, respectively. A higher prevalence of Listeria contaminated samples was found in samples taken in the evening with 0%, 25%, 76% and 100% of samples positive at sampling points 1,2, 3 and 4, respectively. The most frequently recovered species of Listeria isolated from both sets of samples were L. innocua, L. monocytogenes, L. grayi and L. seeligeri (Table 2). The total aerobic mesophilic and psychrophilic counts, together with levels of Enterobacteriaceae and E. coli in pre-processing scald tank water samples were low at 3.28, 2.98, 0.13, and 0.30 log10 cfu/ml, respectively (Table 3). Counts of indicator microorganisms in the scald water samples increased significantly following the passage of flocks through the tank (Table 3). These increases were all found to be statistically significant for both morning and evening samples following the processing of birds when compared with pre-production levels (Po0:05). With the exception of Enterobacteriaceae, counts of indicator
Table 2 Prevalence of Listeria recovered from neck skin samples taken on 10 morning and 5 evening visits to a commercial broiler slaughter plant Sampling Point
No. samples Listeria positivea SP 1 After defeathering SP 2 After final carcase washing SP 3 In-line chill exit SP 4 Final chill exit Listeria species isolatedb L. monocytogenes L. innocua L. grayi L. seeligeri
Sampling time AM
PM
5/50 (10)
0/25 (0)
20/50 (40)
5/25 (20)
28/50 (56)
19/25 (76)
36/50 (72)
25/25 (100)
3/50 (6) 43/50 (86) 3/50 (6) 1/50 (2)
10/31 (32.3) 17/31 (54.8) 4/31 (12.9) 0/31 (0)
a
Results expressed as number of positive isolates/number of samples tested and (%) isolation from neck skin samples obtained from each sampling point. b The prevalence of Listeria spp. recovered from samples is expressed as the no. of specific isolates/total no. Listeria isolates obained after 24 and 48 h incubation on selective media and (%).
organisms in both the morning and evening post-load samples did not alter significantly when the mean numbers of birds processed increased from 3912 to 42 475, respectively (Po0:05).
4. Discussion The current study was carried out in order to assess changes in the microbial populations that may occur during the principal stages of commercial broiler
ARTICLE IN PRESS P. Whyte et al. / Food Microbiology 21 (2004) 59–65 Table 3 Summary of mean counts of total mesophilic and psychrophilic aerobes, Enterobacteriaceae and E. coli recovered from scald tank water samples in the broiler slaughter plant TVC 37 C
TVC 22 C
Enterobacteriaceae E. coli
PS (AM) 3.28 (0.99) 2.98 (0.54) 0.13 (0.29) PL (AM) 5.62 (0.42)a 5.31 (0.42)a 3.87 (0.35)a PL (PM) 5.99 (0.53)a 5.26 (0.17)a 4.93 (0.69)b
0.30 (0.38) 3.63 (0.27)a 3.86 (0.31)a
n ¼ 10 for morning (AM) and 5 for evening (PM) samples. PS=pre-start up in plant, PL=following processing of flocks. PL values with different superscripts are statistically different to corresponding PS values (Po0:05). Counts expressed as mean log10 cfu/ml. PL (AM)=3912 was mean number of birds processed. PL (PM)=42 475 was mean number of birds processed. ( )=standard deviation.
processing. The investigation examined the extent of bacterial contamination which carcasses acquired during slaughter and dressing procedures. Our study also evaluated whether the levels of microbial contaminants on carcasses was dependent on the time of day that processed carcasses were sampled. 4.1. Effect of sampling point and time The aerobic mesophilic and psychrophilic counts observed in the plant on both morning and evening visits exhibited a similar trend with a reduction in total numbers on carcasses observed between post-plucking and post-evisceration stages. The reductions recorded were statistically significant for samples collected on evening visits only (Po0:05). Total aerobic counts did not subsequently change significantly in samples from post-evisceration to the exit from in-line chilling. Similar trends were reported by Salvat et al. (1993), who found TVC counts of 5.22, 4.98 and 5.09 log10 cfu/g on neck skin samples recovered after plucking, evisceration and carcass dressing in air chilled processing plants. Clouser et al. (1995a) observed significant decreases in total mesophilic counts from breast skin samples when postplucking and post-chilling stages were compared. The enumeration of Enterobacteriaceae from neck skin samples in the plant during this study revealed different trends between samples taken in the morning and evening. Counts in samples recovered during morning visits increased significantly following evisceration and chilling (Po0:05). These findings were in agreement with those of Lillard (1989) who reported an increase in Enterobacteriaceae counts when post-plucking and post-evisceration stages were compared. James et al. (1992) observed a decrease in numbers when preevisceration and post-chill samples were compared and attributed this to the use of an immersion chilling system. Enterobacteriaceae counts recovered from evening samples decreased following evisceration and
63
increased after initial chilling, with a small decrease observed on exit from the final chill. The Enterobacteriaceae counts for both sampling times were not statistically different between sampling points or over time. E. coli levels for both morning and evening visits increased generally through all four sampling points. The trend observed in the present study concurred with Abu-Ruwaida et al. (1994) who reported an increase in E. coli counts following evisceration. However, a reduction in counts on carcasses was reported by the same authors after air chilling; this is in contrast to the findings of this study. James et al. (1993) noted a reduction in E. coli counts when pre-evisceration and pre-chill carcasses were compared. Levels of S. aureus decreased significantly following evisceration and final carcass washing (Po0:05). No further significant changes in counts were observed in post-evisceration samples taken on both morning and evening visits. Salvat et al. (1993) reported similar results to those observed in this investigation with reductions from 2.44 to 2.18 log10 cfu/g between postplucking and evisceration stages, respectively. AbuRuwaida et al. (1994) noted less significant decreases during processing than those reported in the current study. 4.2. Microbiology of scald water samples Scald tank water samples were examined both prior to production start-up and following the processing of all bird loads sampled. The total aerobic mesophilic and psychrophilic counts, together with levels of Enterobacteriaceae and E. coli in pre-production samples were low at 3.28, 2.98, 0.13, and 0.30 log10 cfu/ml, respectively. This demonstrated that an effective overnight water replacement and cleaning protocol was in place. Schuler and Badenhop (1972) also confirmed low levels of total aerobic counts in pre-production scald water samples. No statistical difference in microbial counts was observed in samples taken after processing for morning and evening samples, with the exception of Enterobacteriaceae (Po0:05). Levels of Enterobacteriaceae recovered from scald water samples was similar to those reported by Lillard (1990), while Abu-Ruwaida et al. (1994) isolated higher numbers of total aerobes. The mean number of birds passed through the scald tank prior to samples being taken on morning and evening visits was 3912 and 42 475, respectively. The observed microbiological profile of the tank water samples suggested that the number of bacteria entering the tank via contaminated birds equilibrated relatively quickly with the number of organisms killed as a result of the water temperature of 52 C. It would appear, therefore, that scald tank water becomes rapidly contaminated with organisms of faecal origin following the commencement
ARTICLE IN PRESS 64
P. Whyte et al. / Food Microbiology 21 (2004) 59–65
of daily processing and that the levels do not continuously increase during the subsequent passage of birds through the tank. However, it must be noted that the risk of cross-contamination occurring by pathogens such as Salmonella and Campylobacter during scalding increases as birds from subsequent flocks are processed during the day (Genigeorgis et al., 1986; Oosterom et al., 1983). 4.3. Recovery of Listeria spp. The isolation of Listeria from neck skin samples during the present investigation revealed consistent increases through the various stages of processing, where ascending prevalences were observed between Sampling Points 1, 2, 3 and 4 for both morning and evening samples (Table 2). Also, a higher prevalence of Listeria was detected on evening samples; this suggested the increased occurrence of cross-contamination within the plant over time. As Listeria can form biofilms on solid stainless steel surfaces, cross-contamination from equipment to carcasses may have resulted in higher prevalences being observed over time. In addition, these Listeria biofilms have been demonstrated to be more resistant to sanitation or disinfection than other microbial contaminants so that a background level of the organism may have been present on equipment surfaces even following overnight plant and equipment cleaning. The high recovery rates observed in this study were in agreement with Franco et al. (1995) and Capita et al. (2001), who reported rates of 96% and 95% in broiler carcasses, respectively. The prevalence of L. monocytogenes also increased over time, with evening samples accounting for 32.2% of isolates, compared to 6% from morning samples. L. innocua was the most prevalent species recovered on both morning and evening samples, with 86% and 54.8% isolated, respectively. Bailey et al. (1989) reported the presence of L. monocytogenes on 23% of broiler carcasses sampled, with an overall Listeria spp. isolation rate of 38%, while Gitter (1976) recovered the organism from 14.7% of broiler carcasses.
5. Conclusions Overall, an interesting finding of the current study was the apparent decrease in bacterial load on broiler carcasses slaughtered and dressed in the plant over time, with counts on evening samples consistently lower than those found on equivalent morning samples. A similar trend has also been reported by other workers (McNab et al., 1991; Renwick et al., 1993). The latter authors did not identify possible reasons for their findings, suggesting that further investigation is warranted. It is possible that these variations are principally dependent on the microbiological profile of flocks or loads of live birds
presented for slaughter. Therefore, the farm of origin or conditions during live bird transportation for example, may be more significant factors in determining the levels of indicator organisms on processed carcasses rather than the slaughter and dressing procedures. The microbiological data accumulated from the broiler processing plant enables a number of conclusions to be made. In general, microbial loads were reduced on skin samples during processing (Table 2). However, in terms of end product safety, E. coli counts increased on samples taken following evisceration on both morning and evening visits. This demonstrated that carcasses were exposed to faecal material during evisceration, and consequently potentially pathogenic micro-organisms, during processing and that existing carcass washing procedures were incapable of restoring counts to preevisceration levels. Furthermore, high prevalences of Listeria spp. found on dressed carcasses indicates that significant cross-contamination occurs during typical processing procedures. Finally, the study demonstrated that good hygienic practices in the plant should be observed at all times and incorporated as part of an effective HACCP system. Proper hygiene should minimize exposure of carcasses to faecal material and potential pathogens and reduce opportunities for cross-contamination to occur.
References Abu-Ruwaida, A.S., Sawaya, W.N., Dashti, B.H., Murad, M., Al-Othman, H.A., 1994. Microbiological quality of broilers during processing in a modern commercial slaughterhouse in Kuwait. J. Food Prot. 57, 887–892. Al-Mohizea, I.S., Mashhadi, A.S., Fawwal, A., Al-Shalhat, A., 1994. Microbiological and shelf life assessment of chilled eviscerated whole chicken broilers in Saudi Arabia. Br. Poult. Sci. 35, 519–526. Anonymous, 1994. Enumeration of Escherichia coli in reference materials for food microbiology, BCR/Food Trial 6, in Report 281008005. National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands, pp. 35–39. Anonymous, 2000. European Commission. Opinion of the scientific committee on veterinary measures relating to public health on foodborne zoonoses. Health and Consumer Protection Directorate-General, Brussels. http://www.europa.eu.int/comm/food/fs/sc/ scv/out32en.pdf Anonymous, 2002. Control of Campylobacter species in the food chain. Food Safety Authority of Ireland Report, FSAI, ISBN 1-904465-00-5, pp. 1–42. Bailey, J.S., Fletcher, D.L., Cox, N.A., 1989. Recovery and serotype distribution of Listeria monocytogenes from broiler chickens in the Southeastern United States. J. Food Prot. 52, 148–150. Brown, M.H., Baird-Parker, A.C., 1982. The microbiological examination of meat. In: Brown, M.H. (Ed.), Meat Microbiology. Applied Science Publishers, London, pp. 423–520. Bryan, F.L., 1988. Risks associated with practices, procedures and processes that lead to outbreaks of foodborne diseases. J. Food Prot. 51, 663–673. Capita, R., Alonso-Calleja, C., Moreno, B., Garcia-Fernandez, M.C., 2001. Occurrence of Listeria species in retail poultry meat and
ARTICLE IN PRESS P. Whyte et al. / Food Microbiology 21 (2004) 59–65 comparison of a cultural/immunoassay for their detection. Int. J. Food Microbiol. 65, 75–82. Carson, M.O., Lillard, H.S., Hamdy, M.K., 1987. Transfer of firmly attached 32P-Salmonella typhimurium from raw poultry skin to other surfaces. J. Food Prot. 50, 327–329. Clouser, C.S., Doores, S., Mast, M.G., Knabel, S.J., 1995a. The role of defeathering in the contamination of turkey skin by Salmonella species and Listeria monocytogenes. Poult. Sci. 74, 723–731. Clouser, C.S., Knabel, S.J., Mast, M.G., Doores, S., 1995b. Effect of type of defeathering system on Salmonella cross-contamination during commercial processing. Poult. Sci. 74, 732–741. Fitzgerald, M., Bonner, C., Foley, B., Wall, P.G., 2001. Analysis of outbreaks of infectious intestinal disease in Ireland: 1998 and 1999. Irish Med. J. 94, 140–144. Franco, C.M., Quinto, E.J., Fente, C., Rodriguez-Otero, J.L., Dominguez, L., Cepeda, A., 1995. Determination of the principle sources of Listeria spp. contamination in poultry meat and a poultry processing plant. J. Food Prot. 58, 1320–1325. Genigeorgis, C., Hassuneh, M., Collins, P., 1986. Campylobacter jejuni infection on poultry farms and its effect on poultry meat contamination during slaughtering. J. Food Prot. 49, 895–903. Gitter, M., 1976. Listeria monocytogenes in oven-ready poultry. Vet. Rec. 99, 336. Goepfert, J.M., 1976. The aerobic plate count, coliform and Escherichia coli content of raw ground beef at retail level. J. Food Prot. 39, 175–178. Jacobs-Reitsma, W.F., 1997. Aspects of epidemiology of Campylobacter in poultry. Vet. Quart. 19, 113–117. Jacobs-Reitsma, W.F., 2000. Campylobacter in the food supply. In: Nachamkin, I., Blaser, M.J. (Eds.), Campylobacter, 2nd Edition. American Society for Microbiology, Washington DC, pp. 467–481. James, W.O., Williams, O., Prucha, J.C., Johnston, R., Christensen, W., 1992. Profile of selected bacterial counts and Salmonella prevalence on raw poultry in a poultry slaughter establishment. J. Am. Vet. Med. Assoc. 200, 57–59. James, W.O., Prucha, J.C., Brewer, R.L., 1993. Cost-effective techniques to control human enteropathogens on fresh poultry. Poult. Sci. 72, 1174–1176. Jones, F.T., Axtell, R.C., Rives, D.V., Scheideler, S.E., Tarver, F.R., Walker, R.L., Wineland, M.J., 1991. A survey of Campylobacter jejuni contamination in modern broiler production and processing systems. J. Food Prot. 54, 259–262. Lillard, H.S., 1990. The impact of commercial processing procedures on the bacterial contamination and cross-contamination of broiler carcases. J. Food Prot. 53, 202–204. Lillard, H.S., 1989. Incidence and recovery of salmonellae and other bacteria from commercially processed poultry carcasses at selected pre-and post evisceration steps. J. Food Prot. 52, 88–91. Lillard, H.S., Cox, N.A., Bailey, J.S., Thomson, J.E., 1984. Comparison of brands of media for isolating bacteria from poultry, beef and shrimp. J. Food Prot. 47, 394–397. Mc Nab, W.B., Forsberg, C.M., Clarke, R.C., 1991. Application of an automated hydrophobic grid membrane filter interpreter system at a poultry abattoir. J. Food Prot. 54, 619–622.
65
Mead, G.C., Hudson, W.R., Hinton, M.H., 1993. Microbiological survey of five poultry processing plants in the UK. Br. Poult. Sci. 34, 497–503. Oosterom, J., Notermans, S., Karman, H., Engels, G.B., 1983. Origin and prevalence of Campylobacter jejuni in poultry processing. J. Food Prot. 46, 339–344. Panisello, P.J., Rooney, R., Quantick, P.C., Stanwell-Smith, R., 2000. Application of foodborne disease outbreak data in the development and maintenance of HACCP systems. Int. J. Food Microbiol. 59, 221–234. Renwick, S.A., Mc Nab, B.W., Lowman, H.R., Clarke, R.C., 1993. Variability and determinants of carcass bacterial load at a poultry abattoir. J. Food Prot. 56, 694–699. Roberts, D., Hooper, W., Greenwood, M., 1995. Isolation and enrichment of microorganisms. In: Roberts, D. (Ed.), Practical Food Microbiology, 2nd Edition. Public Health Laboratory Service, London, pp. 130–142. Salvat, G., Allo, J.C., Colin, P. (1993). Evolution of microbiological contamination of poultry carcases during slaughtering: a survey on 12 French abattoirs. In: Proceedings of the 11th European Symposium on Poultry Meat Quality, Tours, France, 4–8 October, pp. 562–568. Schuler, G.A., Badenhop, A.F., 1972. Microbiology survey of equipment in selected poultry processing plants. Poult. Sci. 51, 830–835. Silliker, J.H., Gabis, D.A., 1976. ICMSF method studies. VII. Indicator tests as substitutes for direct testing of dried foods and feeds for Salmonella. Can. J. Microbiol. 22, 971–974. Smoot, M.L., Pierson, M.D., 1997. Indicator microorganisms and microbiological criteria. In: Doyle, M.P., Beuchat, L.R., Montville, T.J. (Eds.), Food Microbiology Fundamentals and Frontiers. ASM Press, Washington DC, pp. 66–80. Thatcher, F.S., Clark, D.S., 1968. Indicator organisms. In: Thatcher, F.S., Clark, D.S. (Eds.), Microorganisms in Foods—Their Significance and Methods of Enumeration. University of Toronto Press, pp. 23–37. Upton, M., 1996. Relationships between pathogenic growth and the general microbiota on raw and processed meat and poultry. In: Sheridan, J.J., Buchanan, R.L., Montville, T.J. (Eds.), HACCP: An Integrated Approach to Assuring the Microbiological Safety of Meat and Poultry. Food and Nutrition Press, Connecticut, pp. 139–150. Uyttendaele, M., De Troy, P., Debevere, J., 1999. Incidence of Salmonella, Campylobacter jejuni, Campylobacter coli, and Listeria monocytogenes in poultry carcasses and different types of poultry products for sale on the Belgian retail market. J. Food Prot. 62, 735–740. Waldroup, A.L., Rathgeber, B.M., Forsythe, R.H., Smoot, L., 1992. Effects of six modifications on the incidence and levels of spoilage and pathogenic organisms on commercially processed post chill broilers. J. Appl. Poult. Res. 1, 226–234. White, P.L., Baker, A.R., James, W.O., 1996. Strategies to control Salmonella and Campylobacter in raw poultry products. Rev. Sci. Tech. L’Office Int. Epizooties 16, 525–541.
www.elsevier.nl/locate/jnlabr/yfmic
The effect of sampling time on the levels of micro-organisms recovered from broiler carcasses in a commercial slaughter plant P. Whytea,*, K. McGilla, C. Monahanb, J.D. Collinsa a
Veterinary Public Health and Food Safety Laboratory, Department of Large Animal Clinical Studies, Faculty of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland b Dawn Farm Foods Ltd., Naas Co., Kildare, Ireland Received 14 November 2002; accepted 10 March 2003
Abstract Changes in the levels of indicator micro-organisms and Listeria on broiler carcasses were determined at various stages of commercial broiler slaughter and processing. In addition, the effect of time of day that sampling was carried out within the plant on the microbial profile of samples was evaluated. Counts of total aerobic organisms (37 C and 22 C), Enterobacteriaceae, E. coli and Staphylococcus aureus on broiler skin samples were consistently lower on samples obtained during evening visits to the co-operating broiler slaughter plant when compared with corresponding morning samples. Counts of S. aureus were significantly lower for both morning and evening samples taken following final carcass rinsing when compared to pre-evisceration equivalents (Po0:05). The number of total viable aerobic bacteria, Enterobacteriaceae and E. coli in scald tank water samples taken early each morning prior to processing start-up were significantly lower than corresponding samples obtained after flocks had been processed where an average of 3912 and 42 475 birds had been processed on morning and evening visits, respectively (Po0:05). The prevalence of Listeria on broiler skin samples consistently increased for both morning and evening samples through the various stages of processing. For example, the prevalence on samples taken in the morning following defeathering, final carcass rinsing, in-line chilling and final carcass chilling was 10%, 40%, 56% and 72%, respectively. The most frequently recovered species of Listeria from these samples were L. innocua (86%), L. monocytogenes (6%), L. grayi (6%) and L. seeligeri (2%). r 2003 Elsevier Ltd. All rights reserved. Keywords: Poultry microbiology; Indicator organisms; Enterobacteriaceae; Escherichia coli; Listeria; Staphylococcus
1. Introduction The handling and consumption of poultry meat is recognized as a major cause of foodborne illness in humans globally (Anonymous, 2000; Fitzgerald et al., 2001), particularly when eaten raw, undercooked or recontaminated and stored following cooking (Bryan, 1988; White et al., 1996). Processed broiler carcasses and raw poultry products are frequently contaminated with pathogenic micro-organisms of public health significance including, Salmonella (Panisello et al., 2000), thermophilic campylobacters (Jacobs-Reitsma, 1997, 2000; Anonymous, 2002), and Listeria (Capita et al., 2001; Uyttendaele et al., 1999). *Corresponding author. Tel.: +353-1-7166074; fax: +353-17166090. E-mail address: [email protected] (P. Whyte). 0740-0020/03/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0740-0020(03)00040-6
The microbiological status of processed poultry carcasses has been reported to be dependent on several key factors, namely: the level of contamination from live birds, numbers and genera of pathogenic or indicator organisms introduced at pre-harvest phases, and the extent of occurrence of contamination and cross-contamination during processing (Abu-Ruwaida et al., 1994). The control of contamination during slaughter and processing has been identified as critical in relation to the prevalence of pathogenic micro-organisms on end products (Mead et al., 1993). Clouser et al. (1995a) identified transport crates, scald water, picker fingers, evisceration equipment, chill water and personnel as potential sources of cross-contamination during slaughter. Cross-contamination can also occur during rearing, transport and holding together with the principal processing stages (Clouser et al., 1995b).
ARTICLE IN PRESS 60
P. Whyte et al. / Food Microbiology 21 (2004) 59–65
Upton (1996) noted that the extent to which contamination occurred on carcasses and the composition of the resulting flora reflected the hygiene standards at processing level, together with the initial microbiological status of the animal. Smoot and Pierson (1997) regarded the application of microbiological criteria using indicator organisms as useful for foods such as poultry which were repeatedly implicated in foodborne disease outbreaks, as the presence of certain organisms indicated the possibility of occurrence of microbiological hazards. However, the limitations associated with the use of indicator organisms in poultry and meat processing as a means of assessment of food safety have been previously documented by Goepfert (1976). The potential microbiological hazards in meat products cannot be assessed nor demonstrated reliably with the use of acceptable levels based on total counts or indicator organisms (Brown and Baird-Parker, 1982). However, the analysis of foods of animal origin for the presence or levels of generic Escherichia coli has been widely accepted as an indicator of faecal contamination, while failure to detect this organism cannot guarantee the absence of other enteric pathogens (Silliker and Gabis, 1976; Brown and Baird-Parker, 1982). The levels of Enterobacteriaceae on poultry carcasses have also been routinely used as a means to indicate inadequate or unhygienic processing or inappropriate handling or storage conditions which may result in the proliferation of a wide range of pathogenic and toxinproducing bacteria, including Salmonella (Brown and Baird-Parker, 1982; Roberts et al., 1995). Total aerobic counts have been recommended as a useful tool in the microbiological assessment of food safety, as high mesophilic counts (25–37 C) may indicate unsatisfactory sanitation or the use of heavily contaminated raw materials (Thatcher and Clark, 1968). Lillard et al. (1984) and Al-Mohizea et al. (1994) also acknowledged the benefit of aerobic plate counts as a criterion in assessing the hygiene of poultry processing plants. The effect of processing techniques on rates of crosscontamination by pathogenic organisms, including, Salmonella and Campylobacter, has been demonstrated by increased prevalences reported following carcass slaughter and dressing procedures (Waldroup et al., 1992; Jones et al., 1991). The mechanisms by which contamination occurs during processing, other than immersion in scald and chill waters, have been described by Schuler and Badenhop (1972) and Carson et al. (1987), who demonstrated carcass–carcass and equipment–carcass contacts to be significant routes in the spread of pathogenic organisms. The present study was carried out to determine the levels of a range of indicator micro-organisms on broiler carcasses sampled at various stages of a commercial
slaughter process. In addition, the study examined whether the microbiological load present on slaughtered broiler carcasses was dependent on the time of day that samples were obtained in the plant. It is frequently assumed that the microbiological profile of broiler carcasses disimproves over time in a processing plant as a result of the build up of high numbers of organisms on equipment surfaces and their dissemination within the plant on to carcasses. However, few studies have been carried out to ascertain and quantify the effect that time of sampling may have on the profile of carcasses processed on the same day within a slaughter plant.
2. Methods 2.1. Description and sampling procedures of the broiler processing plant The slaughter plant used in the current study was part of an integrated broiler operation and processed on average 60 000 birds per day. Processing commenced at 7.30 AM each morning and continued for 12 h or 1.5 shifts. The average throughput ranged from 5000 to 5500 birds per hour. Birds were supplied for slaughter by contract growers to the plant at ages which varied between 35 and 54 days depending on carcass weight requirements. In total, 15 visits to the slaughter plant were made over a 4-month period. Sampling was carried out on the first load of birds of each days throughput for the first 10 visits with samples from the remaining five visits taken from loads processed 7–8 h after daily production had commenced. During each visit, five neck skin samples were aseptically collected from different carcasses at four separate sampling points within the plant. Samples were collected each day from carcasses within the same flock at each sampling point. The sampling points used for the current study were: (i) immediately following defeathering at the transfer point to the evisceration line, (ii) after evisceration and final carcass washing, (iii) following in-line primary air chilling and (iv) after exiting the blast freezing tunnel (final carcass chilling). The equipment used in the plant was specifically designed for commercial broiler processing (Stork, N.V., Naarden, The Netherlands). Carcass temperatures entering the in-line primary air chill were slightly lower than those observed in live birds and averaged 39 C. Carcasses were air chilled in this system for approximately 45–60 min with temperatures of exiting carcasses ranging from +2 C to +8 C depending on mean carcass weights. The temperature of carcasses entering the freezing tunnel averaged +4 C while those exiting the chill dropped to Ca. 2 C. In addition, 100 ml scald water samples were taken each morning from the tank at the point where birds exited each morning prior to the
ARTICLE IN PRESS P. Whyte et al. / Food Microbiology 21 (2004) 59–65
commencement of production and again after the load sampled had been slaughtered. The scald tank used was a single stage unit operating at a temperature of 52 C. All samples were transported to the laboratory at p+4 C and microbiological analysis was carried out on the day of sampling. 2.2. Microbiological analysis For neck skins, 25 g of each sample was aseptically weighed and placed in sterile stomacher bags to which 225 ml of 0.1% peptone water was added. The samples were homogenized for 1 min each using a stomacher (Lab Blender 400 series, Seward Medical, UK). Serial 10-fold dilutions were then prepared by transferring 1 ml of this solution to 9 ml tubes of 0.1% peptone water. Once samples had been stomached and 10-fold dilutions prepared, the levels of total aerobic mesophilic (37 C) and psychrophilic (22 C) organisms, Enterobacteriaceae, E. coli and Staphylococcus aureus and the prevalence of Listeria spp. was established. For total aerobic and Enterobacteriaceae counts, 1 ml of the relevant dilutions were placed in duplicate plates and approximately 20 ml of molten standard plate count agar (Oxoid, Basingstoke, UK) or violet red bile glucose agar (Oxoid, Basingstoke, UK) were added after which the dilutions and molten agar were mixed and allowed to solidify. Total count plates were incubated for 48 and 72 h at 37 C and 22 C, respectively, before colonies were counted. Violet-red bile glucose agar plates were incubated at 37 C for 18–24 h after which characteristic purple haloed colonies were counted. E. coli were enumerated from stomached samples using a method previously described (Anonymous, 1994). Briefly, 0.5 ml of each dilution was spread in duplicate on surface mounted membrane filters (Gelman Sciences, MN, USA) which were placed on the surface of tryptone bile agar plates (Oxoid, Basinstoke, UK). The plates were then incubated overnight at 44 C. After incubation, the membrane filters were removed from the agar plates and placed on equivalent sized filter papers (Whatman International, Maidstone, UK) that had been soaked in 2 ml volumes of indole detection reagent (5% pdimethylaminobenzaldehyde dissolved in 1 N HCl) (Sigma-Aldrich, Dorset, UK). Indole positive colonies developed a pink-purple coloration on the membrane filter surfaces within 20 min and were presumptively counted as E. coli. and S. aureus were presumptively enumerated on the basis of their appearance on BairdParker medium (Oxoid, Basingstoke, UK). Briefly, 0.1 ml volumes of relevant dilutions were spread on duplicate plates which were then incubated for 48 h at 37 C. A sample of 10–15 colonies from each sampling day’s plates were confirmed as coagulase-positive S. aureus using a latex agglutination test (Murex Diagnostics, Kent, UK). Listeria spp. were isolated from
61
samples by adding 50 ml of the stomached sample to an equal volume of double strength pre-enrichment broth (Oxoid, Basingstoke, UK) and incubating for 18–24 h at 30 C. The samples were then selectively enriched by inoculating 0.1 ml volumes of the pre-enrichment media into broths containing a Listeria selective supplement (Oxoid, Basinstoke, UK). Samples were subcultured onto Oxford formulation Listeria selective plates (Oxoid, Basingstoke, UK) which were incubated at 37 C for 24 and 48 h. The confirmation and identification of suspect Listeria colonies on the solid media was then carried out by transferring to tryptone soya agar (Oxoid, Basingstoke, UK) and incubating for 24 h at 37 C. Using Henrys’ illumination technique of holding the plates at a 45 angle to a light source, typical Listeria colonies exhibited a blue to blue-grey colour. Speciation was carried out using the API biochemical profiling system (Biomerieux, Lyon, France). Scald tank water samples were also analysed for total viable counts, Enterobacteriaceae and E. coli levels by making up 10-fold serial dilutions and plating out onto the various selective and differential media as described above. 2.3. Statistical analysis All microbial counts obtained from neck skin samples were transformed to log10 values for subsequent data analysis. Unpaired Student’s t-tests were used to compare counts from both neck skin and scald tank water samples. Significance was defined at the 95% level (Pp0:05). The ‘‘StatView 5’’ programme (SAS Institute Inc., USA) was used for all statistical analyses.
3. Results The levels of all of the indicator organisms enumerated from neck skins were consistently lower on samples obtained during evening visits than those from morning samplings (Table 1). Significantly lower counts of total mesophilic and psychrophilic organisms were observed in evening samples at the final carcass wash, in-line chill exit and final chill exit sampling points when compared with corresponding morning samples (Po0:05). The mean number of Enterobacteriaceae was higher but not statistically significant, on evening samples taken following in-line chilling than in equivalent samples taken during morning visits with 3.79 and 3.37 log10 cfu/g, respectively. Similarly, significant differences in the levels of E. coli and S. aureus were observed between morning and evening samples taken immediately following final carcass washing (Po0:05). Interestingly, levels of Enterobacteriaceae, E. coli and S. aureus found on either sample sets did not statistically differ to finished product sampled exiting the final air chill.
ARTICLE IN PRESS P. Whyte et al. / Food Microbiology 21 (2004) 59–65
62
Table 1 Summary of results of mean total viable mesophilic and psychrophilic aerobes, Enterobacteriaceae, Escherichia coli and Staphylococcus aureus counts from neck skin samples taken during 10 morning and 5 evening visits to the broiler processing plant Sampling time
Total viable counts
SP 1 After defeathering SP 2 After final carcase washing SP 3 In-line chill exit SP 4 Final chill exit
Enterobacteriaceae
E. coli
S. aureus
37 C AM
37 C PM
22 C AM
22 C PM
AM
PM
AM
PM
AM
PM
5.27 (0.27) 4.87a (0.30) 5.12a (0.44) 5.08a (0.26)
5.01* (0.36) 4.44b** (0.61) 4.47b** (0.26) 4.58b** (0.26)
5.05 (0.24) 4.79a (0.32) 5.12a (0.43) 5.15a (0.29)
4.88* (0.41) 4.38b** (0.60) 4.46b** (0.37) 4.64b*,** (0.09)
3.28* (0.39) 3.37* (0.44) 3.79** (0.30) 3.50*,** (0.39)
3.25 (0.28) 3.17 (0.19) 3.37 (0.13) 3.33 (0.15)
3.01 (0.56) 3.26a (0.43) 3.35 (0.48) 3.28 (0.63)
2.90 (0.21) 2.95b (0.35) 3.11 (0.23) 3.20 (0.31)
3.04* (0.56) 2.48a** (0.43) 2.48** (0.48) 2.30** (0.63)
2.89* (0.47) 2.09b** (0.21) 2.40** (0.30) 2.35** (0.28)
n ¼ 50 for morning sampling points (AM), n ¼ 25 for evening sampling points (PM). Counts expressed as log10 cfu/g of mean values. ( )=standard deviation. *Denotes significance in counts between sampling points for each organism category taken during morning visits (Po0.05). **Denotes significance in counts between sampling points for each organism category taken during evening visits (Po0.05). a Denotes significance of morning counts for each organism category taken at the same sampling point (Po0.05). b Denotes significance of afternoon counts for each organism category taken at the same sampling point (Po0.05).
When counts on neck skins obtained during evening visits were examined, it was noted that significant reductions were observed between the defeathering and carcass washing stages for total mesophilic and psychrophilic counts (Po0:05), This trend was also observed between these sampling points in the plant for S. aureus counts on both morning and evening samples. The recovery of Listeria from neck skin samples obtained both during the morning and evening consistently increased through the various stages of processing, with ascending prevalences observed between sampling points 1, 2, 3 and 4, respectively (Table 2). The number of positive samples recovered during morning visits increased from 5% to 72% for sampling points 1 and 4, respectively. A higher prevalence of Listeria contaminated samples was found in samples taken in the evening with 0%, 25%, 76% and 100% of samples positive at sampling points 1,2, 3 and 4, respectively. The most frequently recovered species of Listeria isolated from both sets of samples were L. innocua, L. monocytogenes, L. grayi and L. seeligeri (Table 2). The total aerobic mesophilic and psychrophilic counts, together with levels of Enterobacteriaceae and E. coli in pre-processing scald tank water samples were low at 3.28, 2.98, 0.13, and 0.30 log10 cfu/ml, respectively (Table 3). Counts of indicator microorganisms in the scald water samples increased significantly following the passage of flocks through the tank (Table 3). These increases were all found to be statistically significant for both morning and evening samples following the processing of birds when compared with pre-production levels (Po0:05). With the exception of Enterobacteriaceae, counts of indicator
Table 2 Prevalence of Listeria recovered from neck skin samples taken on 10 morning and 5 evening visits to a commercial broiler slaughter plant Sampling Point
No. samples Listeria positivea SP 1 After defeathering SP 2 After final carcase washing SP 3 In-line chill exit SP 4 Final chill exit Listeria species isolatedb L. monocytogenes L. innocua L. grayi L. seeligeri
Sampling time AM
PM
5/50 (10)
0/25 (0)
20/50 (40)
5/25 (20)
28/50 (56)
19/25 (76)
36/50 (72)
25/25 (100)
3/50 (6) 43/50 (86) 3/50 (6) 1/50 (2)
10/31 (32.3) 17/31 (54.8) 4/31 (12.9) 0/31 (0)
a
Results expressed as number of positive isolates/number of samples tested and (%) isolation from neck skin samples obtained from each sampling point. b The prevalence of Listeria spp. recovered from samples is expressed as the no. of specific isolates/total no. Listeria isolates obained after 24 and 48 h incubation on selective media and (%).
organisms in both the morning and evening post-load samples did not alter significantly when the mean numbers of birds processed increased from 3912 to 42 475, respectively (Po0:05).
4. Discussion The current study was carried out in order to assess changes in the microbial populations that may occur during the principal stages of commercial broiler
ARTICLE IN PRESS P. Whyte et al. / Food Microbiology 21 (2004) 59–65 Table 3 Summary of mean counts of total mesophilic and psychrophilic aerobes, Enterobacteriaceae and E. coli recovered from scald tank water samples in the broiler slaughter plant TVC 37 C
TVC 22 C
Enterobacteriaceae E. coli
PS (AM) 3.28 (0.99) 2.98 (0.54) 0.13 (0.29) PL (AM) 5.62 (0.42)a 5.31 (0.42)a 3.87 (0.35)a PL (PM) 5.99 (0.53)a 5.26 (0.17)a 4.93 (0.69)b
0.30 (0.38) 3.63 (0.27)a 3.86 (0.31)a
n ¼ 10 for morning (AM) and 5 for evening (PM) samples. PS=pre-start up in plant, PL=following processing of flocks. PL values with different superscripts are statistically different to corresponding PS values (Po0:05). Counts expressed as mean log10 cfu/ml. PL (AM)=3912 was mean number of birds processed. PL (PM)=42 475 was mean number of birds processed. ( )=standard deviation.
processing. The investigation examined the extent of bacterial contamination which carcasses acquired during slaughter and dressing procedures. Our study also evaluated whether the levels of microbial contaminants on carcasses was dependent on the time of day that processed carcasses were sampled. 4.1. Effect of sampling point and time The aerobic mesophilic and psychrophilic counts observed in the plant on both morning and evening visits exhibited a similar trend with a reduction in total numbers on carcasses observed between post-plucking and post-evisceration stages. The reductions recorded were statistically significant for samples collected on evening visits only (Po0:05). Total aerobic counts did not subsequently change significantly in samples from post-evisceration to the exit from in-line chilling. Similar trends were reported by Salvat et al. (1993), who found TVC counts of 5.22, 4.98 and 5.09 log10 cfu/g on neck skin samples recovered after plucking, evisceration and carcass dressing in air chilled processing plants. Clouser et al. (1995a) observed significant decreases in total mesophilic counts from breast skin samples when postplucking and post-chilling stages were compared. The enumeration of Enterobacteriaceae from neck skin samples in the plant during this study revealed different trends between samples taken in the morning and evening. Counts in samples recovered during morning visits increased significantly following evisceration and chilling (Po0:05). These findings were in agreement with those of Lillard (1989) who reported an increase in Enterobacteriaceae counts when post-plucking and post-evisceration stages were compared. James et al. (1992) observed a decrease in numbers when preevisceration and post-chill samples were compared and attributed this to the use of an immersion chilling system. Enterobacteriaceae counts recovered from evening samples decreased following evisceration and
63
increased after initial chilling, with a small decrease observed on exit from the final chill. The Enterobacteriaceae counts for both sampling times were not statistically different between sampling points or over time. E. coli levels for both morning and evening visits increased generally through all four sampling points. The trend observed in the present study concurred with Abu-Ruwaida et al. (1994) who reported an increase in E. coli counts following evisceration. However, a reduction in counts on carcasses was reported by the same authors after air chilling; this is in contrast to the findings of this study. James et al. (1993) noted a reduction in E. coli counts when pre-evisceration and pre-chill carcasses were compared. Levels of S. aureus decreased significantly following evisceration and final carcass washing (Po0:05). No further significant changes in counts were observed in post-evisceration samples taken on both morning and evening visits. Salvat et al. (1993) reported similar results to those observed in this investigation with reductions from 2.44 to 2.18 log10 cfu/g between postplucking and evisceration stages, respectively. AbuRuwaida et al. (1994) noted less significant decreases during processing than those reported in the current study. 4.2. Microbiology of scald water samples Scald tank water samples were examined both prior to production start-up and following the processing of all bird loads sampled. The total aerobic mesophilic and psychrophilic counts, together with levels of Enterobacteriaceae and E. coli in pre-production samples were low at 3.28, 2.98, 0.13, and 0.30 log10 cfu/ml, respectively. This demonstrated that an effective overnight water replacement and cleaning protocol was in place. Schuler and Badenhop (1972) also confirmed low levels of total aerobic counts in pre-production scald water samples. No statistical difference in microbial counts was observed in samples taken after processing for morning and evening samples, with the exception of Enterobacteriaceae (Po0:05). Levels of Enterobacteriaceae recovered from scald water samples was similar to those reported by Lillard (1990), while Abu-Ruwaida et al. (1994) isolated higher numbers of total aerobes. The mean number of birds passed through the scald tank prior to samples being taken on morning and evening visits was 3912 and 42 475, respectively. The observed microbiological profile of the tank water samples suggested that the number of bacteria entering the tank via contaminated birds equilibrated relatively quickly with the number of organisms killed as a result of the water temperature of 52 C. It would appear, therefore, that scald tank water becomes rapidly contaminated with organisms of faecal origin following the commencement
ARTICLE IN PRESS 64
P. Whyte et al. / Food Microbiology 21 (2004) 59–65
of daily processing and that the levels do not continuously increase during the subsequent passage of birds through the tank. However, it must be noted that the risk of cross-contamination occurring by pathogens such as Salmonella and Campylobacter during scalding increases as birds from subsequent flocks are processed during the day (Genigeorgis et al., 1986; Oosterom et al., 1983). 4.3. Recovery of Listeria spp. The isolation of Listeria from neck skin samples during the present investigation revealed consistent increases through the various stages of processing, where ascending prevalences were observed between Sampling Points 1, 2, 3 and 4 for both morning and evening samples (Table 2). Also, a higher prevalence of Listeria was detected on evening samples; this suggested the increased occurrence of cross-contamination within the plant over time. As Listeria can form biofilms on solid stainless steel surfaces, cross-contamination from equipment to carcasses may have resulted in higher prevalences being observed over time. In addition, these Listeria biofilms have been demonstrated to be more resistant to sanitation or disinfection than other microbial contaminants so that a background level of the organism may have been present on equipment surfaces even following overnight plant and equipment cleaning. The high recovery rates observed in this study were in agreement with Franco et al. (1995) and Capita et al. (2001), who reported rates of 96% and 95% in broiler carcasses, respectively. The prevalence of L. monocytogenes also increased over time, with evening samples accounting for 32.2% of isolates, compared to 6% from morning samples. L. innocua was the most prevalent species recovered on both morning and evening samples, with 86% and 54.8% isolated, respectively. Bailey et al. (1989) reported the presence of L. monocytogenes on 23% of broiler carcasses sampled, with an overall Listeria spp. isolation rate of 38%, while Gitter (1976) recovered the organism from 14.7% of broiler carcasses.
5. Conclusions Overall, an interesting finding of the current study was the apparent decrease in bacterial load on broiler carcasses slaughtered and dressed in the plant over time, with counts on evening samples consistently lower than those found on equivalent morning samples. A similar trend has also been reported by other workers (McNab et al., 1991; Renwick et al., 1993). The latter authors did not identify possible reasons for their findings, suggesting that further investigation is warranted. It is possible that these variations are principally dependent on the microbiological profile of flocks or loads of live birds
presented for slaughter. Therefore, the farm of origin or conditions during live bird transportation for example, may be more significant factors in determining the levels of indicator organisms on processed carcasses rather than the slaughter and dressing procedures. The microbiological data accumulated from the broiler processing plant enables a number of conclusions to be made. In general, microbial loads were reduced on skin samples during processing (Table 2). However, in terms of end product safety, E. coli counts increased on samples taken following evisceration on both morning and evening visits. This demonstrated that carcasses were exposed to faecal material during evisceration, and consequently potentially pathogenic micro-organisms, during processing and that existing carcass washing procedures were incapable of restoring counts to preevisceration levels. Furthermore, high prevalences of Listeria spp. found on dressed carcasses indicates that significant cross-contamination occurs during typical processing procedures. Finally, the study demonstrated that good hygienic practices in the plant should be observed at all times and incorporated as part of an effective HACCP system. Proper hygiene should minimize exposure of carcasses to faecal material and potential pathogens and reduce opportunities for cross-contamination to occur.
References Abu-Ruwaida, A.S., Sawaya, W.N., Dashti, B.H., Murad, M., Al-Othman, H.A., 1994. Microbiological quality of broilers during processing in a modern commercial slaughterhouse in Kuwait. J. Food Prot. 57, 887–892. Al-Mohizea, I.S., Mashhadi, A.S., Fawwal, A., Al-Shalhat, A., 1994. Microbiological and shelf life assessment of chilled eviscerated whole chicken broilers in Saudi Arabia. Br. Poult. Sci. 35, 519–526. Anonymous, 1994. Enumeration of Escherichia coli in reference materials for food microbiology, BCR/Food Trial 6, in Report 281008005. National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands, pp. 35–39. Anonymous, 2000. European Commission. Opinion of the scientific committee on veterinary measures relating to public health on foodborne zoonoses. Health and Consumer Protection Directorate-General, Brussels. http://www.europa.eu.int/comm/food/fs/sc/ scv/out32en.pdf Anonymous, 2002. Control of Campylobacter species in the food chain. Food Safety Authority of Ireland Report, FSAI, ISBN 1-904465-00-5, pp. 1–42. Bailey, J.S., Fletcher, D.L., Cox, N.A., 1989. Recovery and serotype distribution of Listeria monocytogenes from broiler chickens in the Southeastern United States. J. Food Prot. 52, 148–150. Brown, M.H., Baird-Parker, A.C., 1982. The microbiological examination of meat. In: Brown, M.H. (Ed.), Meat Microbiology. Applied Science Publishers, London, pp. 423–520. Bryan, F.L., 1988. Risks associated with practices, procedures and processes that lead to outbreaks of foodborne diseases. J. Food Prot. 51, 663–673. Capita, R., Alonso-Calleja, C., Moreno, B., Garcia-Fernandez, M.C., 2001. Occurrence of Listeria species in retail poultry meat and
ARTICLE IN PRESS P. Whyte et al. / Food Microbiology 21 (2004) 59–65 comparison of a cultural/immunoassay for their detection. Int. J. Food Microbiol. 65, 75–82. Carson, M.O., Lillard, H.S., Hamdy, M.K., 1987. Transfer of firmly attached 32P-Salmonella typhimurium from raw poultry skin to other surfaces. J. Food Prot. 50, 327–329. Clouser, C.S., Doores, S., Mast, M.G., Knabel, S.J., 1995a. The role of defeathering in the contamination of turkey skin by Salmonella species and Listeria monocytogenes. Poult. Sci. 74, 723–731. Clouser, C.S., Knabel, S.J., Mast, M.G., Doores, S., 1995b. Effect of type of defeathering system on Salmonella cross-contamination during commercial processing. Poult. Sci. 74, 732–741. Fitzgerald, M., Bonner, C., Foley, B., Wall, P.G., 2001. Analysis of outbreaks of infectious intestinal disease in Ireland: 1998 and 1999. Irish Med. J. 94, 140–144. Franco, C.M., Quinto, E.J., Fente, C., Rodriguez-Otero, J.L., Dominguez, L., Cepeda, A., 1995. Determination of the principle sources of Listeria spp. contamination in poultry meat and a poultry processing plant. J. Food Prot. 58, 1320–1325. Genigeorgis, C., Hassuneh, M., Collins, P., 1986. Campylobacter jejuni infection on poultry farms and its effect on poultry meat contamination during slaughtering. J. Food Prot. 49, 895–903. Gitter, M., 1976. Listeria monocytogenes in oven-ready poultry. Vet. Rec. 99, 336. Goepfert, J.M., 1976. The aerobic plate count, coliform and Escherichia coli content of raw ground beef at retail level. J. Food Prot. 39, 175–178. Jacobs-Reitsma, W.F., 1997. Aspects of epidemiology of Campylobacter in poultry. Vet. Quart. 19, 113–117. Jacobs-Reitsma, W.F., 2000. Campylobacter in the food supply. In: Nachamkin, I., Blaser, M.J. (Eds.), Campylobacter, 2nd Edition. American Society for Microbiology, Washington DC, pp. 467–481. James, W.O., Williams, O., Prucha, J.C., Johnston, R., Christensen, W., 1992. Profile of selected bacterial counts and Salmonella prevalence on raw poultry in a poultry slaughter establishment. J. Am. Vet. Med. Assoc. 200, 57–59. James, W.O., Prucha, J.C., Brewer, R.L., 1993. Cost-effective techniques to control human enteropathogens on fresh poultry. Poult. Sci. 72, 1174–1176. Jones, F.T., Axtell, R.C., Rives, D.V., Scheideler, S.E., Tarver, F.R., Walker, R.L., Wineland, M.J., 1991. A survey of Campylobacter jejuni contamination in modern broiler production and processing systems. J. Food Prot. 54, 259–262. Lillard, H.S., 1990. The impact of commercial processing procedures on the bacterial contamination and cross-contamination of broiler carcases. J. Food Prot. 53, 202–204. Lillard, H.S., 1989. Incidence and recovery of salmonellae and other bacteria from commercially processed poultry carcasses at selected pre-and post evisceration steps. J. Food Prot. 52, 88–91. Lillard, H.S., Cox, N.A., Bailey, J.S., Thomson, J.E., 1984. Comparison of brands of media for isolating bacteria from poultry, beef and shrimp. J. Food Prot. 47, 394–397. Mc Nab, W.B., Forsberg, C.M., Clarke, R.C., 1991. Application of an automated hydrophobic grid membrane filter interpreter system at a poultry abattoir. J. Food Prot. 54, 619–622.
65
Mead, G.C., Hudson, W.R., Hinton, M.H., 1993. Microbiological survey of five poultry processing plants in the UK. Br. Poult. Sci. 34, 497–503. Oosterom, J., Notermans, S., Karman, H., Engels, G.B., 1983. Origin and prevalence of Campylobacter jejuni in poultry processing. J. Food Prot. 46, 339–344. Panisello, P.J., Rooney, R., Quantick, P.C., Stanwell-Smith, R., 2000. Application of foodborne disease outbreak data in the development and maintenance of HACCP systems. Int. J. Food Microbiol. 59, 221–234. Renwick, S.A., Mc Nab, B.W., Lowman, H.R., Clarke, R.C., 1993. Variability and determinants of carcass bacterial load at a poultry abattoir. J. Food Prot. 56, 694–699. Roberts, D., Hooper, W., Greenwood, M., 1995. Isolation and enrichment of microorganisms. In: Roberts, D. (Ed.), Practical Food Microbiology, 2nd Edition. Public Health Laboratory Service, London, pp. 130–142. Salvat, G., Allo, J.C., Colin, P. (1993). Evolution of microbiological contamination of poultry carcases during slaughtering: a survey on 12 French abattoirs. In: Proceedings of the 11th European Symposium on Poultry Meat Quality, Tours, France, 4–8 October, pp. 562–568. Schuler, G.A., Badenhop, A.F., 1972. Microbiology survey of equipment in selected poultry processing plants. Poult. Sci. 51, 830–835. Silliker, J.H., Gabis, D.A., 1976. ICMSF method studies. VII. Indicator tests as substitutes for direct testing of dried foods and feeds for Salmonella. Can. J. Microbiol. 22, 971–974. Smoot, M.L., Pierson, M.D., 1997. Indicator microorganisms and microbiological criteria. In: Doyle, M.P., Beuchat, L.R., Montville, T.J. (Eds.), Food Microbiology Fundamentals and Frontiers. ASM Press, Washington DC, pp. 66–80. Thatcher, F.S., Clark, D.S., 1968. Indicator organisms. In: Thatcher, F.S., Clark, D.S. (Eds.), Microorganisms in Foods—Their Significance and Methods of Enumeration. University of Toronto Press, pp. 23–37. Upton, M., 1996. Relationships between pathogenic growth and the general microbiota on raw and processed meat and poultry. In: Sheridan, J.J., Buchanan, R.L., Montville, T.J. (Eds.), HACCP: An Integrated Approach to Assuring the Microbiological Safety of Meat and Poultry. Food and Nutrition Press, Connecticut, pp. 139–150. Uyttendaele, M., De Troy, P., Debevere, J., 1999. Incidence of Salmonella, Campylobacter jejuni, Campylobacter coli, and Listeria monocytogenes in poultry carcasses and different types of poultry products for sale on the Belgian retail market. J. Food Prot. 62, 735–740. Waldroup, A.L., Rathgeber, B.M., Forsythe, R.H., Smoot, L., 1992. Effects of six modifications on the incidence and levels of spoilage and pathogenic organisms on commercially processed post chill broilers. J. Appl. Poult. Res. 1, 226–234. White, P.L., Baker, A.R., James, W.O., 1996. Strategies to control Salmonella and Campylobacter in raw poultry products. Rev. Sci. Tech. L’Office Int. Epizooties 16, 525–541.