The potential use of chilling to control the growth of Enterobacteriaceae on porcine carcasses and the incidence of E. coli O157:H7 in pigs
Mary Lenahan, Ashtown Food Research Centre, Ashtown, Dublin 15, Ireland. E-mail: email@example.com
Aims: To (i) monitor the presence of Enterobacteriaceae as indicators of faecal contamination on pig carcasses, (ii) examine the potential use of chilling as a critical control point (CCP) and establish its influence on pig carcass categorization by Decision 471/EC and (iii) determine the incidence of E. coli O157:H7 in pigs.
Methods and Results: Porcine faecal samples and carcass swabs were collected before and after chilling at four Irish pig abattoirs and examined for Enterobacteriaceae and E. coli O157:H7. Chilling generally reduced Enterobacteriaceae counts on carcasses, but increases were also observed, particularly in one abattoir. E. coli O157:H7 was absent from carcasses before chilling, present on 0·21% after chilling and was recovered from 0·63% of faecal samples. All of the isolates were found to contain virulence genes associated with clinical illness in humans.
Conclusions: The data show that overall chilling had the capacity to reduce the numbers of carcasses positive for the presence of Enterobacteriaceae.
Significance and Impact of Study: The influence of chilling on the categorization of pig carcasses suggests that it has the potential to improve the numbers of acceptable carcasses and the process could be used as a CCP within a HACCP plan.
In EC Commission Decision 2001/471/EC and Commission Regulation 2073/2005, the Enterobacteriaceae are used as indicators of faecal contamination of fresh meat carcasses (Anonymous 2001;Anonymous 2005). The performance criteria for the mean Enterobacteriaceae counts (log10CFU cm−2) on pigs are: acceptable <2·0, marginal 2·0–3·0 and unacceptable >3·0 (Anonymous 2001, 2005). These criteria are applied to carcasses at the end of the slaughter line and before chilling, as it is intended that they be used to demonstrate the efficacy of the slaughter process only. There may however be an advantage in assessing the presence of these indicators after chilling, as this process has the potential to reduce their presence on pig carcasses and is therefore a better measure of the actual numbers of these organisms on carcasses prior to retail sale.
Chilling is used successfully as a critical control point (CCP) in slaughter plants in Australia based on the efficacy of the chilling process to reduce the numbers of E. coli on carcass surfaces (Anonymous 2004). Meat companies are required to validate their carcass-chill procedures to comply with the Australian Meat Standard AS 4696 : 2007 (Anonymous 2007). However, this is not the case in the European Union (EU) and in practice, the capabilities of chillers and chill-carcass cycles vary from plant to plant (Sheridan 2004). The basis of HACCP (Hazard Analysis and Critical Control Point) in relation to fresh meat is that the numbers of pathogenic organisms on carcass surfaces are reduced or their growth is prevented as elimination is not possible (Sheridan 2000). Within this definition, chilling has the potential to accomplish these two objectives, but the refrigeration conditions must be shown to be capable of achieving them. As pathogenic bacteria are infrequently present on carcass surfaces, generic E. coli and other Enterobacteriaceae are used to indicate the potential presence of enteric pathogens. As already outlined above, the indicator used in the EU is the Enterobacteriaceae and in this study, the behaviour of these organisms during chilling was monitored.
E. coli O157:H7 causes a wide range of clinical symptoms in humans including haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS) (Griffin and Tauxe 1991). Ruminants are regarded as the main reservoir of E. coli O157:H7 and the pathogens have been recovered from both Irish cattle (McEvoy et al. 2003) and sheep (Lenahan et al. 2007). Research has indicated that while pigs are potential carriers of E. coli O157:H7, the reported incidence is generally much lower than in ruminants (Wasteson 2001).
This study investigated the levels of Enterobacteriaceae on carcasses as indicators of faecal contamination and examined the potential use of chilling as a means of reducing their numbers or controlling their growth. The incidence of E. coli O157:H7 on carcasses before and after chilling and in faeces was also determined, as this is an important human pathogen and no previous work on its prevalence in Irish pigs has been carried out.
Materials and methods
Study design and slaughter process
Four pig abattoirs (A, B, C, and D) were each visited four times over a eleven-month period from January to November 2004. Abattoirs were visited once during each of the four seasons – visit 1 was from January to March, visit 2 from April to July, visit 3 from August to September and visit 4 from October to November. Three of the abattoirs (A, B, C) slaughtered 250 pigs per hour and the fourth (D) 75 pigs per hour. In general, pigs were slaughtered by the following process in the four abattoirs. Animals were held in lairage, stunned and immediately exsanguinated by sticking before dressing. They were scalded at 61°C (+ 2°C) for approx. 8 min, dehaired, ejected onto a gambrelling table and hoisted onto an overhead conveyor rail. The carcasses were passed through a singer, operating at approx. 1200°C for 15 s, polished and transferred into a separate evisceration area. Carcasses were debunged by detaching the rectum with a knife, which was then enclosed in a plastic bag to limit faecal leakage during subsequent processing. Carcasses were eviscerated, split from hind to fore using a manual splitting saw, and the head and spinal cord removed. Finally, carcasses were trimmed, weighed, graded, spray washed and chilled overnight.
At each visit, carcass hygiene levels were assessed by collecting 30 carcass swabs before chilling and 30 swabs after chilling, from different sides of the same animal, to determine Enterobacteriaceae numbers. The presence of E. coli O157:H7 was investigated by taking 30 whole animal body swabs before chilling and whole body swabs from 30 different animals after chilling at each visit. In addition, 30 faecal samples were collected and examined for the presence of the pathogen. In total, the Enterobacteriaceae were enumerated on 960 carcasses and the presence of E. coli O157:H7 was determined from 1440 carcass and faecal samples.
Enterobacteriaceae numbers were enumerated by swabbing the lead side or carcass of an animal at the end of the slaughter line before chilling and the opposite trailing side of the same animal 24 h after chilling. Carcass sides (area 6270 cm2) were swabbed using a 150 cm2 polyurethane sponge (Sydney Heath & Son, Stoke-on-Trent, UK) starting from the hindquarter downward to the forequarter (O’Brien et al. 2007).
To determine the presence of E. coli O157:H7, whole body swabs (area 12 540 cm2) were taken from animals before chilling and a different set of animals after chilling. Separate polyurethane sponges (75 cm2) were used to swab the two carcass sides from each animal and these were pooled to give a composite whole body sample (Lenahan et al. 2007).
Faecal samples were obtained during evisceration by aseptically slitting the distal colon with a sterile scalpel and removing faecal material from the colon using a sterile wooden tongue depressor (Medical Supply Co., Dublin, Ireland) (Lenahan et al. 2007). The faecal samples were taken from a different set of animals from those tested for E. coli O157:H7 before and after chilling. All carcass swabs and faecal samples were transported to the laboratory in a cool box containing ice packs and stored overnight in a laboratory chill (1°C) for examination within 24 h.
Enumeration of Enterobacteriaceae
Carcass swabs were examined for the presence of Enterobacteriaceae using the procedure described in ISO 4833. In brief, swabs (10 g) in 90 ml MRD (Oxoid) were stomached in a model 400 stomacher (A.J. Seward, UK) for 30 s at 260 rev min−1 and 1 ml aliquots of the resultant suspension used to make (1 : 10 v/v) serial dilutions in MRD. A 1 ml aliquot of the swab/MRD homogenate and the serial dilutions were pour plated with 15 ml of Violet Red Bile Glucose agar (VRBGA) (Oxoid), allowed to solidify and overlaid with 10 ml VRBGA. Plates were incubated at 37°C for 24 h.
Isolation of E. coli O157:H7
A 90 ml volume of Brilliant Green Bile Broth (BGBB) (Oxoid) was added to each whole body composite sample (10 g) and stomached for 30 s at 260 rev min−1. Sterile cotton-tipped swabs (Nuova Aptaca, Canelli, Italy) used to sample 0·5 g faeces were broken off into 5 ml volumes of BGBB and vortexed for 10 s. The carcass swabs and faeces were selectively enriched in BGBB by incubating at 37°C for 6 h (Feder et al. 2003). One millilitre aliquots of the carcass or faecal enrichment broth were added to 20 μl of immunomagnetic beads coated with anti-E. coli O157 antibody (Dynabeads® anti-E. coli O157; Dynal A.S., Oslo, Norway) and processed according to the manufacturer’s instructions. The bead/sample suspensions were plated in duplicate onto Sorbitol MacConkey agar (SMAC) (Oxoid) supplemented with cefixime-tellurite (CT) (Oxoid) and incubated at 37°C for 24 h. Where present, up to five nonsorbitol fermenting colonies were selected from each duplicate plate and subjected to biochemical confirmation. Colonies from CT-SMAC were streaked onto Nutrient Agar (Oxoid) to obtain a purified culture and incubated for 24 h at 37°C. Colonies from the Nutrient agar were biochemically confirmed by streaking onto Levines Eosin Methylene Blue agar (EMB) (Oxoid), Phenol Red Sorbitol agar (Oxoid) supplemented with 4-methylumbelliferyl-B-d-Glucuronide (Oxoid) (PRS-MUG), inoculated into Tryptone Tryptophane medium (TT) (Oxoid) and all were incubated at 37°C for 24 h. Colonies displaying a green metallic sheen on EMB, no fluorescence on PRS-MUG when illuminated with UV light, and which were indole positive in TT medium on addition of Kovacs reagent (Oxoid), were examined using a E. coli O157:H7 latex agglutination kit (Wellcolex, Basingstoke, UK) according to the manufacturer’s instructions to check for the presence of the O157 antigen. All presumptive E. coli O157 isolates were stored at −20°C on ProtectTM Stock Culture Beads (Technical Service Consultants Ltd, Heywood, UK) to enable PCR analysis to be carried out at a later date.
Detection of virulence genes
All presumptive E. coli O157 isolates were subjected to PCR analysis to confirm the presence of the O157 antigen and H7 flagellum and to determine presence or absence of 11 other genes that encode proteins considered to have a role in clinical illness. Porcine isolates stored at −20°C on ProtectTM Stock Culture Beads were resuscitated by incubating one bead in 10 ml Brain Heart Infusion broth (BHI) (Oxoid) at 37°C for 18 h. After incubation, genomic DNA was extracted from a 1 ml portion of bacterial cells in BHI using a DNeasy blood & tissue kit (QiagenTM, Crawley, UK) as per the manufacturer’s instructions for gram-negative bacteria. For each assay, 5 μl of genomic DNA was used in each 50 μl PCR reaction. PCR primer sequences and reaction conditions were obtained from previously published work on the genes for O157, vt1, vt2, eaeA, hlyA (Paton and Paton 1998), H7 (Fratamico et al. 2000), tir (Higgins et al. 2003), espA, espB, espP (McNally et al. 2001), espF (Crane et al. 2001), etpD (Schmidt et al. 1999) and katP (Brunder et al. 1996). An E. coli O157:H7 isolate, ATCC 43895, obtained from the Central Meat Control Laboratory, Dept. of Agriculture, Abbotstown, Dublin, Ireland was included as a positive control in each PCR assay while sterile-filtered, DNase & RNase free water (Sigma-Aldrich, Munich, Germany) was used as a negative control. Isolates confirmed as E. coli O157:H7 by PCR were phage typed at the Laboratory of Enteric Pathogens, Centre of Infections, 61 Colindale Avenue, London NW9 5HT, UK.
Statistical analysis of Enterobacteriaceae counts
The experiment was designed as a split-split-plot with abattoirs (A, B, C, D), visits (1, 2, 3, 4) and stages (before and after chilling) on the main plots, sub-plots and sub-sub-plots respectively. The limit of detection for Enterobacteriaceae counts was 0·002 CFU cm−2 (−2·70 log10CFU cm−2). Zero counts were recorded for carcasses when Enterobacteriaceae numbers were below the limit of detection. To cater for zero values, all bacterial counts were increased by 0·001 prior to applying a log transformation to the data and analysis of variance was carried out using Genstat (VSN International, Hemel Hemprtead, UK). When the results were tested for normality by the Anderson-Darling, Cramer-von Mises and Watson tests, the analysis indicated that the data were not log normally distributed (Genstat) at the 1% level.
In consequence, the data were analysed using the nonparametric Mann–Whitney and Kruskal–Wallis tests. The former test was used to compare before and after chilling at each visit for individual abattoirs. The latter test was used to compare the four abattoirs at each visit before and after chilling. The analyses were performed using Genstat. Subsequent to the Kruskal–Wallis test, comparisons between individual abattoirs were made using a multiple comparison test (Conover 1980).
Using the Mann–Whitney test, it was established that before and after chilling there were significant differences (P < 0·05) in carcass counts at each visit for individual abattoirs (Table 1). Exceptions were noted in relation to carcass counts at abattoir C during visits two and four and at abattoir A during visit three, where carcass counts were not statistically different. When carcass counts were compared using the Kruskal–Wallis test, significant differences were observed before chilling between abattoirs during all visits (Table 1). Counts on carcasses in abattoir A were similar to those in abattoirs B, C and D during visits one, two and four, while abattoirs B and C were similar during visits two and four. When carcass counts were compared after chilling, significant differences were observed between abattoirs during all visits. It was noted that at visit two, there were no differences between carcass counts at abattoir C and the remaining three abattoirs.
Table 1. Enterobacteriaceae counts (mean log10CFU cm−2) on pig carcasses. Individual abattoirs at each visit were compared before and after chilling using the Mann–Whitney test. The four abattoirs at each visit were compared (i) before and (ii) after chilling using the Kruskal–Wallis test
To determine the initial levels of contamination on carcasses and the exact influence of chilling on the Enterobacteriaceae counts on carcasses, the data from each of the 30 carcasses swabbed before and after chilling during each visit in the four abattoirs were examined. The numbers of carcasses showing reductions, increases or unchanged counts after chilling are recorded in Table 2. These data are not intended to show statistical differences between Enterobacteriaceae counts on carcasses before and after chilling, but demonstrate changes in counts resulting from the chilling process on a carcass-by-carcass basis.
Table 2. The influence of chilling in relation to reductions (R), increases (I), or no changes (NC) in Enterobacteriaceae counts (mean log10CFU cm−2) on pig carcasses in four abattoirs during four visits
|NC||2||0·75||0·75|| 4||0·23||0·23|| 1||0·90||0·90|| 0||–||–|
|I||7||0·60||0·99|| 5||0·12||0·37||10||0·03||1·23|| 6||0·30||1·10|
|NC||7||nd*||nd|| 3||nd||nd|| 4||−0·08||−0·08|| 3||nd||nd|
|I||8||−1·05||−0·30|| 0||–||–|| 0||–||–|| 2||0·45||1·35|
|NC||14||nd||nd|| 2||nd||nd||12||nd||nd|| 1||nd||nd|
|I||17||−0·18||0·55|| 5||−0·04||0·21||16||nd||1·63|| 8||1·01||2·03|
|NC||9||−0·07||−0·07|| 6||−0·1||−0·1|| 7||−0·04||−0·04|| 1||nd||nd|
The highest mean initial counts were almost exclusively (7/16) on carcasses in the four abattoirs during visit 1 and abattoir D during all visits, indicating a much higher level of contamination on these carcasses. Reductions in counts as a result of chilling were recorded in all abattoirs and from all visits. When considered across abattoirs, the highest reductions were in abattoir D and the lowest in abattoir C. The data in Table 2 also clearly demonstrate that in abattoir C, while there were reductions in counts, increases also occurred after chilling during three of the four visits.
The Enterobacteriaceae are used to assess the hygienic status of fresh meat carcasses, using the performance criteria set down in EC Commission Decision 2001/ 471/EC and Commission Regulation 2073/2005, which assigns carcasses to one of three categories of contamination based on mean counts (log10CFU cm−2) of acceptable <2·0, marginal 2·0–3·0 and unacceptable >3·0 (Anonymous 2001, 2005). Using these criteria, the numbers of acceptable, marginal and unacceptable carcasses before and after chilling are shown in Table 3. Before chilling, a majority of carcasses at all abattoirs and visits were found to be acceptable, except for abattoir D. In abattoir D, at each visit, there were fewer acceptable and more marginal and unacceptable carcasses compared with the other abattoirs indicating that carcass hygiene was poorer at this plant. This result is related to the data in Table 2, which show a higher level of contamination on carcasses in this abattoir, before chilling and after chilling.
Table 3. Categorization of pig carcasses as a result of chilling
Chilling increased the numbers of acceptable carcasses at abattoirs A, B and D, but not in abattoir C during visits 1, 2 and 4. The numbers of marginal and unacceptable carcasses were also reduced by chilling, in particular, in abattoir D. After chilling, in abattoir C, the numbers of acceptable carcasses were reduced during visits 1, 2 and 4, as were the numbers of marginal carcasses during these visits. In addition, the numbers of unacceptable carcasses increased during visit 1. These data suggest that carcass contamination levels increased during chilling in abattoir C, a result noted previously in Table 2.
E. coli O157:H7
The survey detected E. coli O157:H7 in 0·63% (3/480) of faecal samples and only 0·21% (1/480) on carcasses after chilling. All samples found to contain E. coli O157:H7 were taken during the third quarter of the sampling period from August to September 2004. Of the four isolates recovered, two were found in faecal samples taken during the same visit to abattoir A. The other two isolates recovered from a faecal sample and from an after chill carcass swab were isolated during the same visit to abattoir C (Table 4). DNA from the E. coli O157:H7 isolates was extracted and characterized using PCR analysis. Although genetic profiles were established for a maximum of ten nonsorbitol fermenting isolates from CT-SMAC per sample (i.e. up to five per duplicate plate when colonies present), a single genetic profile was found to represent all isolates from each positive sample. Analysis by PCR established that the faecal E. coli O157:H7 isolates (n = 3) carried all the genes investigated in this study, while the carcass isolate (n = 1) contained less than half the genes detected in the faecal samples (Table 4). All faecal isolates possessed the genes for both verotoxins, while the carcass isolate carried only the gene for verotoxin 1. Both E. coli O157:H7 isolates from abattoir A were phage type PT 8, while the faecal isolate from abattoir C was phage type PT 32. The carcass isolate found after chilling at abattoir C had an unidentifiable phage type.
Table 4. Phage types and presence (+) or absence (−) of virulence genes in E. coli O157:H7 isolated from porcine faeces and after chill carcass
|17 Aug 2004||Faeces||A||PT 8||+||+||+||+||+||+||+||+||+||+||+||+||+|
|17 Aug 2004||Faeces||A||PT 8||+||+||+||+||+||+||+||+||+||+||+||+||+|
|02 Sept 2004||Faeces||C||PT 32||+||+||+||+||+||+||+||+||+||+||+||+||+|
|02 Sept 2004||After chill carcass||C||Unidentifiable||+||+||+||−||+||+||−||−||−||−||−||−||−|
Examination of the data using analysis of variance revealed that the Enterobacteriaceae were not log normally distributed over the surface of carcasses and in consequence, it was not possible to compare mean log values statistically. Lack of a normal distribution of these organisms on fresh meat carcasses has been encountered previously (Gill 2000; Gill and Jones 2000). However, nonparametric analysis revealed that there were significant differences (P < 0·05) in carcass counts before and after chilling and between abattoirs. Chilling generally decreased the numbers of Enterobacteriaceae positive carcasses and when the abattoirs were considered over the four visits, these varied from 93% to 59% before chilling, to 68–30% after chilling. These data show that overall chilling had the capacity to reduce the numbers of carcasses positive for the presence of Enterobacteriaceae. The implications of this for the categorization of carcasses in Decision 471/EC will be discussed below.
In terms of Enterobacteriaceae counts, this study recorded reductions during all visits in all abattoirs. Previous studies have noted reductions (Chang et al. 2003; Nesbakken et al. 2008), increases (Bolton et al. 2002) or no change (Gill and Jones 1997) in the numbers of coliforms and E. coli on pig carcasses due to the chilling process and these variations are attributed to differences in chilling parameters between plants (Sheridan 2000). The carcasses in this study were cooled in conventional chillers at all four abattoirs, but the chilling parameters of temperature, carcass spacing, air velocity and relative humidity were not measured. For the successful implementation of chilling as a CCP to control the growth of pathogens on carcasses, critical limits need to be established and the effectiveness of the chilling procedure validated at each plant (Gill and Jones 1997; Sheridan 2000; Anonymous 2004, 2007). According to Gill (2005), the proper control of a carcass cooling process requires the definition and subsequent maintenance of operating parameters that ensure at least the containment and preferably the reduction in the numbers of E. coli on carcasses.
It was observed that there were increases in counts after chilling in abattoir C, which occurred during three of the four visits. The counts at this abattoir increased in such a consistent manner that it suggests growth of these organisms occurred or that contamination during processing was responsible for the observed effect. The possibility of growth was most likely during visit 1 and 4, as the increases were sufficiently high for such an event to have occurred. Under normal chilling conditions (5–6°C), it has been shown that Enterobacteriaceae were not significantly different before and after chilling, which is related to the very long lag and generation times at these temperatures (Newton et al. 1978; Smith 1985). It is assumed therefore to facilitate this level of growth, the temperature in the chill would have been about 12–15°C, indicating a failure in the refrigeration system on that day (Smith 1985). Where smaller increases occurred in this abattoir, it is more difficult to determine whether the observed effect is due to growth or contamination during or after chilling. It is not possible to determine the source of this contamination accurately because the use of a whole carcass swab technique precluded the collection of information from specific sites and whether it occurred before or during chilling. In this experiment, the lead side (carcass) of an animal was swabbed before chilling and the opposite trailing side swabbed separately after chilling. The most plausible explanation, however, is that the carcasses were contaminated just before or during chilling, based on the relatively low numbers of positive carcasses and the low counts before chilling. This could have arisen from aerial sources, handling by personnel during chill loading or from carcasses touching other carcasses during chilling.
The increased counts on carcasses in all abattoirs during visit 1 compared to other visits suggest that there may have been a seasonal effect in the contamination of carcasses. This is not a true seasonal effect, however, because the visits to the abattoirs during this period were carried out on different dates during January, February and March. The higher counts observed during this time may be a reflection of the cleanliness of the animal housing or transport of animals to the abattoir, resulting in the presentation of animals that are dirtier than at other times of the year. As the presence of Enterobacteriaceae on carcasses is synonymous with faecal contamination, it seems reasonable to suggest that the higher levels of contamination with these organisms is associated with increased faeces on the live animal (Heuvelink et al. 2001). A relationship between increased levels of soil on live animals and increased bacterial counts on beef carcasses has been established in the past (McEvoy et al. 2000). The highest levels of carcass contamination were observed in abattoir D during each of the four visits. The increased levels of contamination could not be explained on the basis of geographical, husbandry or lairage issues. The most likely explanation for the increased faecal contamination was from inadequate attention to detail during evisceration, which has been observed previously in another Irish abattoir (Pearce et al. 2004). It has been shown previously that line speed facilitates good manufacturing practices especially during evisceration, which is an important source of faecal contamination (Heuvelink et al. 2001). In this study, however, abattoir D had the slowest line speed (75 pigs per hour) and this factor was therefore unlikely to be responsible for the higher levels of contamination observed.
One of the objectives of this study was to determine the possible influence of chilling on pig carcass categorization in relation to Decision 471/EC. The data showed that in general, chilling improved the numbers of acceptable carcasses in all abattoirs at each visit, with the exception of abattoir C, where the effect of increased counts was clearly evident. This was also the case in relation to the marginal carcasses in abattoirs A and B and unacceptable carcasses in abattoir D. Although the reductions in counts were generally small as a result of chilling, it was apparent that this process could have a significant influence on the categorization of carcasses. An efficient chilling regime, designed to deliver consistent reductions in Enterobacteriaceae counts would seem a worthwhile endeavour, as it could improve carcass safety. This is particularly important because, as discussed elsewhere, the system of corrective action presently described in Decision 471/EC to deal with problems leading to contamination of carcasses during slaughter is unworkable (Hutchison et al. 2005; O’Brien et al. 2007). While not an ideal solution, reducing the numbers of marginal and unacceptable carcasses using chilling could help improve this problem by reducing the need for corrective action.
This study investigated the prevalence of E. coli O157:H7 on carcasses before and after chilling and in addition from faecal samples. The pathogen was recovered from 0·21% (1/480) of carcasses after chilling and 0·63% (3/480) of faecal samples. As the pathogen was detected at such low levels, it was not possible to determine if chilling had any influence on its isolation from carcasses. E. coli O157:H7 has been detected in pig faeces at levels comparable to this study, in America (2%; Feder et al. 2003) and Norway (0·1%; Johnsen et al. 2001). E. coli O157 has also been recovered from pig faeces in the Netherlands (0·1%; Heuvelink et al. 1999) and Great Britain (0·3%; Milnes et al. 2008). E. coli O157:H7 has been recovered in Irish cattle (McEvoy et al. 2003) and sheep (Lenahan et al. 2007), but this is the first record of its presence in pigs. The only report to date of E. coli O157:H7 from a pork product causing clinical illness appears to be from the consumption of a dry fermented traditional salami manufactured from pork meat (Conedera et al. 2007).
PCR analysis detected the vt1, vt2, eaeA and hlyA genes associated with human pathogenesis in all of the faecal E. coli O157:H7 isolates recovered by this study, while the one isolate recovered on an after chill carcass carried the genes for vt1, eaeA and hlyA. The genetic profile of E. coli O157:H7 isolated in this study were typical of those isolated from Irish patients suffering clinical illness and were the same phage types. In 2006, 87% of E. coli O157:H7 strains isolated from Irish clinical patients carried the vt2 gene only, while 13% carried genes for both vt1 and vt2 (Garvey et al. 2008). One of the faecal isolates recovered in our study was PT 32 which was the predominant phage type (47%) in clinical patients in the Republic of Ireland in 2006 and the two remaining faecal isolates recovered were PT 8, which accounted for 11% of the pathogen isolated from Irish clinical patients in that year (Garvey et al. 2008). Molecular characterization of the E. coli O157:H7 isolated from pigs by our study suggests that they may be potentially pathogenic to humans, as their genetic profiles and phage types were similar to those outlined above in relation to clinical infections. However, the incidence of E. coli O157:H7 in pigs was extremely low and they are not considered a primary reservoir for clinical infection in Ireland.
In conclusion, it was important to establish if chilling had the ability to reduce Enterobacteriaceae counts consistently on pig carcasses which would be necessary if it were to be used as a CCP in a pig HACCP plan. The data revealed that reductions occurred on 57% of the carcasses examined, while 17% showed no change. Of considerable importance was the observation that 26% of carcasses showed increased counts. While this observation was related largely to contamination problems suggested in abattoir C, small increases also occurred in the other abattoirs.
Finally, in the EU the Enterobacteriaceae are used as indicators of faecal contamination on carcasses (Anonymous 2001, 2005), but it has been suggested that the use of E. coli as an indicator organism would be more appropriate for this purpose (McEvoy et al. 2004; Stuijk and Mossel 2005; Jordan et al. 2007). The Enterobacteriaceae family comprise a large number of organisms while E. coli are a smaller group more specifically associated with faeces and are therefore better indicators of faecal contamination on carcasses. E. coli are the preferred indicator organism in Australia, the United States and elsewhere (Gill et al. 1996; Sofos et al. 1999; Jordan et al. 2007).
The authors would like to thank Dr Dermot Harrington for performing the statistical analysis on the Enterobacteriaceae results and the Laboratory of Enteric Pathogens, London, UK, for carrying out phage typing of isolates. We acknowledge with gratitude funding from the Department of Agriculture and Food under the Food Institutional Research Measure (FIRM), Project no. 01/R&D/D/170.