Processing practices contributing to Campylobacter contamination in Belgian chicken meat preparations

International Journal of Food Microbiology 128 (2008) 297–303

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o

Processing practices contributing to Campylobacter contamination in Belgian chicken meat preparations Imca Sampers a,b,⁎, Ihab Habib c,e, Dirk Berkvens d, Ann Dumoulin a,b, Lieven De Zutter c, Mieke Uyttendaele a a

Laboratory of Food Microbiology and Food Preservation, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgium Research Group EnBiChem, Department of Industrial Engineering and Technology, University College of West-Flanders, Graaf Karel de Goedelaan 5, B-8500 Kortrijk, Belgium Department of Veterinary Public Health and Food Safety, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium d Department of Animal Health, Unit of Epidemiology and Biostatistics, Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium e Division of Food Hygiene and Control, High Institute of Public Health (HIPH), Alexandria University, 165 El-Horrya Avenue, Alexandria, Egypt b c

a r t i c l e

i n f o

Article history: Received 31 March 2008 Received in revised form 25 August 2008 Accepted 30 August 2008 Keywords: Food Processing practices Campylobacter Chicken meat preparations Freezing Skin

a b s t r a c t The aim of this study was to obtain insight into processing practices in the poultry sector contributing to the variability in Campylobacter contamination in Belgian chicken meat preparations. This was achieved by company profiling of eleven food business operators, in order to evaluate variation of processing management, in addition to statistical modelling of microbiological testing results for Campylobacter spp. contamination in 656 end product samples. Almost half (48%) of chicken meat preparation samples were positive for Campylobacter spp. Results revealed a statistically significant variation in Campylobacter contamination between 11 chicken meat producers across Belgium at both quantitative and qualitative detection levels. All producers provided Campylobacterpositive samples, but prevalence ranged from 9% up to 85% at single producer level. The presence or addition of skin during production of chicken meat preparations resulted in almost 2.2-fold increase in the probability of a sample being positive for Campylobacter, while chicken meat preparations made from frozen meat, or partly containing pre-frozen meat, had a significant (Odds Ratio = 0.41; CI 95% 0.18:0.98) lower probability of being positive for Campylobacter. However, the quantitative results indicated that the positive freezing effect on Campylobacter count was compromised by the presence and/or adding of skin. © 2008 Elsevier B.V. All rights reserved.

1. Introduction As in many other industrialized countries (Moore et al., 2005), Campylobacter is the leading cause of foodborne bacterial gastrointestinal illness in Belgium (Scientific Institute of Public Health, 2007). Epidemiological and molecular studies attribute poultry meat as a major source for sporadic infection and some outbreaks as well (Friedman et al., 2004; Gormley et al., 2007; Kärenlampi et al., 2007; Merchant et al., 2007; Tam et al., 2007). Specifically chicken, also due to the high amount of consumption, is considered as the main risk for C. jejuni (Humphrey et al., 2007; Vellinga and Van Loock, 2002). C. jejuni strains are considered to be sensitive to adverse environmental conditions, which possesses a paradox as to how they survive the stress encountered during food processing and storage (Harrington et al., 2007). Although microbial growth is absent during refrigeration/freezing of chicken meat, C. jejuni has been shown to display physiological activity at 4 °C. Freezing decreases counts of C. jejuni and reductions of 1 to 3 log10 ⁎ Corresponding author. Laboratory of Food Microbiology and Food Preservation, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgium. Tel.: +32 9 264 61 77; fax: +32 9 255 55 10. E-mail address: [email protected] (I. Sampers). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.08.024

in fresh foods stored at −15 to −20 °C have been observed, but C. jejuni does survive during frozen storage in poultry samples (Bhaduri and Cottrell, 2004). Chicken meat preparations ((CMPs) e.g. burgers or sausages of minced meat, marinated or seasoned wings or filets… intended to be eaten) are a new food type introduced in the Belgian market and gaining growing popularity by consumers. They may contain skin due to their nature (e.g. chicken wings) or because of sensory characteristics (e.g. minced meat preparations) (Uyttendaele et al., 2006). All of these items have in common that they have been manipulated extensively during processing and as such also have a potential for (high) Campylobacter contamination levels not only on the surface of the meat but also interior to the meat preparation. As a result there is an increased risk for transfer or survival of Campylobacter not only by cross-contamination but also by undercooking as described by a risk assessment study on chicken meat preparations by Uyttendaele et al. (2006). Previous studies identified certain factors contributing to the risk of Campylobacter contamination at broiler farm level, in the slaughter house or at domestic setting, but to our knowledge few studies (e.g. Iceland study reported by Stern et al., 2003) describe how working practices at meat processing level affect the prevalence of

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Table 1 Classification of companies as big, medium and small, based on their turnover, the proportion of CMP contributing to the turnover, and number of employees

Big Medium Small

Turnover (in million euros)

Proportion of CMP (in million euros)

Number of employees

N20 N10 b10

N2 Between 1 and 2 b1

N 90 N 20 b 20

Campylobacter in the end product. In particular, in order to elaborate HACCP plans in poultry processing industry or to define intervention measures by competent authorities it is important to better understand the behaviour of C. jejuni and C. coli in the food processing environment (Humphrey et al., 2007). To determine the impact of processing practices commonly applied by food business operators (FBO) involved in the production of CMPs on the extent and variability of Campylobacter spp. contamination in this particular food type, a national survey of selected FBO was performed. The present manuscript reports on the processing management profiles of these companies and the statistical analysis of the Campylobacter spp. contamination data in function of the companies' characteristics. This approach was believed to provide better understanding of factors in poultry processing that influence variability in Campylobacter contamination levels in the CMPs, which is an essential element to elaborate a risk based auditing and inspection (International Commission for Microbiological Specification in Foods, 2002). More information in relation to overall prevalence and numbers of Campylobacter present in CMPs and particular related to type of products and seasonal effects is described by Habib et al. (2008). 2. Materials and methods 2.1. Sampling frame The study focused on FBO involved in the production of CMPs. These meat preparations refer to a range of consumer products including portioned, cut chicken meat with intact structure or minced meat, to which spices or other ingredients (seasoning, marinating, coating…) are added to improve sensory properties or texture. The meat preparations are intended to be subjected to heat treatment before consumption (Uyttendaele et al., 2006). Companies were selected from the Belgian Federal Agency for the Safety of the Food

Chain (FASFC) FBO list of 2005. The list was also further updated and verified by consulting collaborators from the poultry unions and the distribution sector. The companies were classified according to their activities: slaughterhouse (S), cutting plant (C) and/or meat preparation/minced meat (M). The 61 companies identified to have activities in the production of CMPs (either M, CM or SCM) were contacted by post and/or electronic mail and by telephone. The company contact package included a) invitation for participation in the national survey explaining its objectives, b) the definition of CMP with examples of food types, c) confidentiality agreement, d) questionnaire about the producer's production flowchart and distribution network. No bias was introduced when a total of 22 out of 61 companies agreed to participate in the survey. Matching the EFSA guidelines on good practices for design of field survey (Anonymous, 2006c), we adopted targeted sampling approach by selecting 11 out of the 22 companies, those represent more than 85% of Belgian product sale of chicken based meat preparations (indeed this was already true by the selection of 3 of the large FBO). The companies were grouped as big, medium and small, based on their turnover, the proportion of CMPs contributing to the turnover, and number of employees (Table 1). The 11 companies represented different variations of production chain; a) from small (n = 3), medium (n = 3) and big operators (n = 5); b) including operators starting from slaughtering, to cutting plant, ending with final product (SCM activities; n = 6); operators starting from cutting plant to final product (CM activities; n = 4), and one operator exclusively focusing on the CMPs processing phase (M activities). All companies have a conventional production, except company J which has an organic production. A 2nd FBO (company A) started an organic production line from April 2007 onwards. Companies delivered their end products either directly to consumer in own retail shops, or to independent butcher shops', grand distribution, or catering. From our contacts with the distribution sector it was confirmed that all major super markets in Belgium sell only CMPs produced in Belgium. The 11 companies included in our study were located across Belgium (Fig. 1). It was however noticed that the majority of the FBO active in the production of CMPs are located in the Flanders region of Belgium (54 of 61 companies). Ten out of 11 companies were selected in the Flanders region. 2.2. Campylobacter data collection During the period from February to December 2007, data with regard to the prevalence of Campylobacter (presence/absence testing) and level of contamination (enumeration) were obtained by

Fig. 1. Geographical distribution of companies involved in the production of chicken meat preparations in Belgium. Numbers inside the map refer to total number of companies (61) per province. Numbers in lateral boxes refer to the actual numbers of companies in the study, selected out of the 22 FBO presented between brackets inside the map.

I. Sampers et al. / International Journal of Food Microbiology 128 (2008) 297–303 Table 2 Distribution of chicken meat preparations samples in the survey (February to December 2007) per company (11 companies) and per product form Company ID

Burgers

Chicken meat preparation type Minced meat

Sausages

Breast

Legs

Wings

A B C D E F G H I J K Total

5 1 7 12 0 11 2 12 3 9 9 11%

7 19 1 5 11 13 7 5 18 11 2 15%

11 5 20 20 14 10 19 17 7 13 10 22%

9 12 2 8 19 10 32 7 20 17 17 23%

17 1 13 24 19 1 10 4 16 24 10 21%

3 0 9 10 7 0 0 0 13 3 3 7%

299

portioned, stuffed or seasoned or marinated or coated, inclusion of prior frozen parts, presence of skin), and if it derived from conventional or organic production.

Total

2.3. Company profiling 8% 6% 8% 12% 11% 7% 11% 7% 12% 12% 8% 100%

randomised sampling, over different months and from different slaughter batches and product forms, at the 11 companies. The sample size was calculated in accordance with the EFSA scientific panels recommendations (Anonymous, 2006d), based on an assumed annual prevalence of approximately 50% (Ghafir et al., 2007), with a desired confidence interval (CI) of 95% and 5% accuracy. Samples per sampling day were chosen to obtain as much as possible CMPs from the 3 product groups (minced meat, portioned with and without skin), and if possible from different batches. Based on the size of the companies, maximum of 6 (from big companies and company J) or 4 (from medium and small companies) samples were taken per visit. Difference in number of samples between the companies is due to the production rate. Table 2 shows that samples were uniformly distributed over companies and product forms during the survey. Chicken wings were mostly delivered by the big companies (C, D, E and I), but also by medium (company A and K) and small (company J) companies. The meat preparations were sampled either from the end of the processing line or from end products stored in cooling cells (maximum storage time 24 h) ready for transport to retailers. Most of the samples (86%) were tested on the day of collection or were kept in the laboratory fridge at 2 °C and tested the following day. Each sample was given a unique reference number. The corresponding sampling form contained next to sampling date and hour, also information of the batch (production date), product type (minced or

Information with regard to processing practices, good manufacturing practices, hygiene procedures and cleaning and disinfection protocols and the implementation of food safety management systems by the individual FBO was collected by completing a check list with the quality manager/production responsible of each company. Information gathered through this initial visit was regularly updated in subsequent visits during sampling. 2.4. Method of analysis Enumeration and detection after enrichment were performed according to the guidelines of the ISO 10272:2006 method (Anonymous, 2006a,b). A representative 10 g test portion was homogenized with 90 ml Bolton enrichment broth (CM0983 plus supplement SR183, Oxoid, UK) and testing was carried out in parallel as follows, (i) direct plating: 1 ml of the initial homogenate (10− 1) was spread plated over four (0.2, 0.3, 0.3, and 0.1 ml) Modified Charcoal Cefoperazon Deoxycholate agar plates (mCCDA) (CM739 plus supplement SR155, Oxoid, UK). From a further serial dilution (10− 2), 0.1 ml was spread plated on mCCDA. Plates were incubated microaerobically (5% CO2, 5% H2, and 85% N2) at 41.5 °C and enumerated after 48 h. (ii) Enrichment culture: 10 ml from the same sample homogenate was transferred to a sterile tube and incubated microaerobically at 41.5 °C, 10 µl was subsequently plated onto mCCDA, and the presence of presumptive Campylobacter growth was checked after 48 h incubation. Multiplex-PCR was used for positive results confirmation and differentiation of C. coli and C. jejuni according to procedures described by Vandamme et al. (1997). 2.5. Statistical analyses Statistical analysis was carried out in Stata SE/10.0 (StataCorp., 2007). A generalised linear model was used with a logarithmic link function and a negative binomial error distribution for the count data and a logit link and binomial error distribution for the binary data (infected–not infected). The design effect (clustering because

Fig. 2. Variability in Campylobacter contamination between the 11 Belgian companies. X-axes show companies identification from A to K (+ whether they are big, medium or small) versus % positive samples with their confidence intervals (95%; estimation of proportions) (n = 656, 11 factories, February to December 2007).

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variation found in the prevalence between the 11 producers is also found in the counts of Campylobacter (Fig. 3). Companies B, C and D with the highest prevalence's, also show significantly higher counts. 3.2. Company profiling on food safety management systems Based on the initial assessment visits to each company, no major differences could be identified with regard to aspects of GMP's and GHP's according to the EU regulation 853/2004. All FBO operate under a food safety management system and have a functional, approved and documented HACCP based system in-place for more than 3 years (except for companies F, H and K their HACCP system is completely or partly certified). Some companies were additionally certified to conform to the British Retail Consortium (BRC) global standard for food safety (companies A, B, C, D and E) or the Dutch HACCP Norm (company I). Fig. 3. Distribution of Campylobacter spp. counts in chicken meat preparations samples from 11 FBO across Belgium. The boxes show values between the 25th and the 75th percentiles. The highest counts in positive samples (below the 10th and over the 90th percentile) are shown as circles in the figure.

companies are the primary sampling unit) was taken into account to ensure that standard errors were computed correctly. 3. Results 3.1. Variation in Campylobacter spp. contamination between companies Overall 48% of chicken meat preparation samples (n = 656) were positive for Campylobacter spp. The average of Campylobacter concentration was 1.68 log10 CFU/g with a standard deviation of ±0.64. Fig. 2 reveals a significant degree of variability in Campylobacter contamination between production companies involved. All producers, organic and conventional production, have provided Campylobacter-positive samples, however with a variation ranging from 9% for producer F to 85% for producer D. Company A, a medium size producer and standing in the middle regarding Campylobacter contamination (35%), served in the analysis as reference in the model. Producer D is by far the most significant (P b 0.0001) FBO among the eleven selected companies in providing Campylobacter-positive samples (Odds Ratio = 10.5; 95% CI 4.6:24.4). Producer B ranked second, with Odds Ratio = 6.1 (95% CI 2.4:15.6), followed by company C with Odds Ratio = 3.9 (95% CI 1.7:8.8). On the other hand, the prevalence of Campylobacter in samples from producer F was significantly lower (P = 0.005) as indicated by an Odds Ratio of 0.18 (95% CI 0.06:0.6). The

3.3. Company profiling on processing practices The bigger companies, running a continuous production line from slaughter over cutting to production of CMPs are using raw non-frozen materials directly from the slaughter and cutting line or after a maximum cooling of 24 h at 4 °C. The complete process may be finished within 4 h. Smaller companies because of logistics choose to have a stock of frozen raw materials (Table 3) and depending on demand use both defrosted raw materials and fresh raw materials to produce the CMPs. Overall, the processing of the meat preparations in comparison to slaughtering and cutting is more manually and not inline or automated. The temperature control is well performed in every company and the adopted working temperatures are according to the Regulation (EC) No 853/2004 of the European Parliament and of the Council of 29 April 2004 (12 °C) or even below. During processing, transport, and storage by the producer, poultry products are held at refrigeration and freezing temperatures for time periods of various durations (Table 3). Table 3 summarises company's protocols with regard to the raw materials used in processing of CMPs and with regard to the pattern of using frozen meat and presentation of skin. The raw material used to prepare minced meat is derived from lean muscles, including adherent fatty tissues. Table 4 shows that 58% (207/354) of the total tested samples with skin were Campylobacter positive; 66% (101/153) of portioned CMPs with skin (marinated or seasoned chicken breast, legs, wings), and 53% (106/201) of minced CMPs with skin (e.g. sausages, burgers) were Campylobacter positive. On the other hand, only 25% (37/149) of CMPs samples (totally or partially) of frozen raw materials were Campylobacter positive. Also, the producer's capacity (big, medium,

Table 3 Composition of minced meat and percentage of frozen chicken meat in the minced meat preparations at the 11 selected companies with time and/or temperature for freezing, thawing, processing and preservation ID

Composition minced meat

% pre-frozen

Freezing time

Thawing temperature and time period

Working temperature

Temperature cooling cell

A B C D E F G

Chicken leg with skin/sometimes filet is added Filet and boned, skinned thigh, skin is added afterwards Boned thigh with skin, without filet Filet and boned, skinned thigh, skin is added afterwards Thigh with skin Pork 10%, chicken 40%, turkey 40%, no skin Boned thigh with skin (for catering; more, frozen skin is added) Boned thigh without skin, no pork Thigh with and without skin Thigh with skin and cutlings Boned thigh without skin and pork (lard), with exception of the product 100% chicken then skin is used

50% 50% 0% 0% 0% 30% Retail: 0%; catering: 45% or 75% 100% 0% 100% 0%

Few days/few weeks Max. 1 month NAa NA NA Thigh max. 3 months Min. 3 weeks; max. 6 months Max. 1 week NA 2–4 weeks NA

2 °C/24 h 12 °C/max. 24 h NA NA NA b4 °C/overnight (12–16 h) −2 °C/max. 24 h

8 °C 12 °C 12 °C 2–5 °C 7 °C 3–7 °C 8 °C

0–2 °C 0–1 °C 0–4 °C 0–2 °C 2–4 °C b 4 °C −2 °C

4 °C NA −8 °C/3 days NA

10–12 °C 10 °C 12 °C 12 °C

b 4 °C 0–1 °C 0 °C 3 °C

H I J K a

Not applicable.

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Table 4 Distribution of Campylobacter-positive chicken meat preparations samples in relation to the use of skin, frozen component, and producers capacity and the logistic regression model of the variables affecting Campylobacter contamination of Belgian chicken meat preparations (n = 656, 11 factories, February to December 2007) Variable

Classes

Number tested

Campylobacter-positive status %

Description

ORa

P-value

95% CIb

Skin

Without skin With skin Not frozen (fresh) Frozen (total or partial) Big Medium Small

302 354 507 149 348 143 165

36 58 55 25 60 48 22

Skin is present (e.g. drumstick or wings), or added to the product (e.g. sausage and burgers) during processing Product is produced from frozen meat, or contains a pre-frozen meat

2.20

0.003

1.41–3.45

0.41

0.046

0.18–0.98

2.69 2.41

0.019 0.127

1.22–5.94 0.74–7.82

0.37 0.89

0.019 0.820

0.31–2.61 1.60–6.42

Freeze Capacity

a b

Production capacity with the small as reference Big Medium Production capacity with the big as reference Small Medium

Odds Ratio. Confidence interval.

small) affects the Campylobacter prevalence, as 60% of samples from big companies were contaminated with Campylobacter compared to 22% for samples obtained from small companies (Table 4). Table 4 describes exposure variables and modelling outputs for the significant impact of the effect of the use of prior frozen raw materials and the inclusion of skin on the variability of Campylobacter contamination in chicken meat preparations. The statistical model indicates that the odds of a sample to be Campylobacter positive increases as the sample contained skin or skin was added during processing. In biological terms, the presence or addition of skin during chicken meat preparations processing resulted in 2.2-fold increase in the probability of a sample being positive for Campylobacter. On the other hand, chicken meat preparations made from frozen meat, or partial addition of pre-frozen meat, had a significantly (Odds Ratio = 0.41; 95% CI 0.18:0.98) lower probability of being positive for Campylobacter. With regard to producer's capacity, samples obtained from big producers resulted in 2.7-fold increase in the odds of being positive for Campylobacter while samples from small producers had a significantly lower probability of being positive. Fig. 4 plots the quantitative effect of the presence of skin and freezing on the counts of Campylobacter. Counts of Campylobacter in samples containing skin, or to which skin was added during processing, had an expected log count of 1.67 more than compared to samples in which skin was stripped before processing (Fig. 4A). On the other hand, Campylobacter concentration in products made from frozen meat (totally or partially) had an expected log count of 1.44 less compared to those made from fresh meat (Fig. 4B). The former findings can be understood more by careful interpretation of interaction between freezing and use of skin (added or already in product). Fig. 5 shows that the positive freezing effect on Campylobacter count was compromised by the presence and/or adding skin. 4. Discussion 4.1. Variability in contamination between producers The design of our survey including a targeted sampling strategy and combining microbiological analysis of end products with company profiling of selected companies involved in the production of chicken meat preparations shows the potential of such approach in the identification of possible processing practices affecting the Campylobacter prevalence. All companies had a good documented and functional HACCP based system in place, achieved positive evaluation from governmental control agency (FASFC) and obtained certification on different commercial standards. Still, a high degree of variability in Campylobacter contamination between 11 FBO involved in the production of CMPs across Belgium at both quantitative and qualitative detection levels has been found. The generalised concept that Campylobacter contamination is high all over the chicken meat

industry should be re-evaluated. Therefore, it is important to realise that each FBO is unique, owing to differences in raw material, logistics, processing practices, plant layout, equipment design and performance, personnel, type of food being produced and other factors. The statistical model output reveals that samples obtained from big producers had a higher probability for being Campylobacter positive, while the opposite was for samples obtained from small producers. This finding does not imply that big producers are bad or that small ones are good, but it should draw the attention to possible processing practices behind such variability. We had a good insight about management and processing procedures within surveyed FBO. Noticeably, big producers were more in a position of supplying big supermarkets chains whom impose a consumer-driven demand for chicken meat preparations made of fresh meats. Therefore, and opposite to the case of small producers, totally or partially frozen meat had a very limited share in chicken meat preparations from big producers. The variability in using fresh versus frozen meat ingredients might be one of the factors explaining contamination variability between big and small businesses. Lower numbers of Campylobacter have been noticed in chicken parts without skin (Berrang et al., 2001). Chicken wings belonging to the group of portioned form products have a higher probability of being positive for Campylobacter because they contain almost exclusively skin. These products were mostly delivered by the big producers. 4.2. Freezing and including skin as variables affecting Campylobacter contamination It is clear that the composition of CMPs and the portion of frozen components vary between companies. Many of the companies add chicken skin for sensory purposes if skin was not already present in the chicken parts used. This variability should be taken into consideration while interpreting variation in Campylobacter contamination between producers. Statistical analysis of test results obtained during the survey showed an influence of the use of partially frozen meat on the one hand and the inclusion of skin on the other hand as determining factors in the overall prevalence of Campylobacter in CMPs. Results presented indicate that total or partial freezing of chicken meat included in products formulation had a positive effect by lowering the odds of a sample being positive for Campylobacter. Ritz et al. (2007) investigated the effect of freezing at −20 °C for Campylobacter on chicken meat (on the skin, below the skin and on the muscle). Their study found 0.9–3.2 log decline in two weeks. Freezing for more than two weeks yielded no additional effect, in agreement with other studies (Bhaduri and Cottrell, 2004; Lee et al., 1998). However, although microbial growth is absent during freezing, and due to reduced water activity (additions of salts, spices) and lowered temperature, a portion of Campylobacter may be killed, a fraction of the Campylobacter population may survive or be sub-lethally injured

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Active and inactive C. jejuni cells show to be located at the bottom of feather follicles and deep channels of chicken skin during storage at 4 °C for 72 h. These sites can provide a suitable microenvironment for the survival of Campylobacter in chicken skin (Chantarapanont et al., 2003). Moreover, protective components, such as proteins, fatty acids, and oils that may be within the surface structure of chicken skin (including follicles) may enhance the survival of Campylobacter by inhibiting the formation of ice crystals. The degree of protection afforded by chicken skin during freezing may not be equivalent to that provided by products containing no skin (Davis and Conner, 2007; Solow et al., 2003). This may account for the difference in recovery of C. jejuni from frozen samples with skin versus frozen without skin concluded in our study. The extreme values in C. jejuni contamination support the conclusions described above. The extreme value of Campylobacter count (3 logs) represented a frozen coated burger with skin; this may also point to the fact that skin can have a role in Campylobacter protection during freezing. On the other hand the three extreme values (4.5, 3.9, 3.8 logs) within products made from not frozen meat and containing skin, were either legs or wings. The extreme value for CMP not frozen and without skin (4.3 logs) was a marinated chicken breast. These are exceptions compared to similar samples and can possibly be explained by cross-contamination. Cross-contamination was possibly also the reason for the two highest values (one chicken leg and the marinated chicken breast), which were from the same sampling day, from the same company, but one with and one without skin. 4.3. Conclusions

Fig. 4. Effect of including skin [A] and freezing [B] on the concentration of Campylobacter in Belgian chicken meat preparations (n = 656, 11 companies, February to December 2007). The dashed lines correspond to the 10 CFU/g (limit of quantification) and 100 CFU/g contamination levels.

(Bhaduri and Cottrell, 2004; Georgsson et al., 2006; Jasson et al., 2007) and still render infective Campylobacter cells. Thus, freezing alone will not add a significant margin of safety with respect to Campylobacter contamination and cannot replace sanitary production and handling (Bhaduri and Cottrell, 2004). Introducing an energy consuming freezing process and frozen storage of raw materials prior to the production of CMPs should be looked at for cost-effectiveness as for the big companies for which the rapid and continuous on line production of CMPs would need to be interrupted as well as an investment for extending frozen storage capacities. Smaller companies are actually already (partially) freezing the raw materials due to logistic reasons. On the other hand, the presence or adding skin during processing of chicken meat preparations was associated with significantly higher Campylobacter counts, and resulted in 2.2-fold increase in the probability of a sample being positive for Campylobacter. Davis and Conner (2007) reported that the incidence on raw, retail poultry products decreases from 76% on whole to 48% on skin-on split breast to only 2% on boneless, skinless breast meat. Campylobacter contamination was low in companies F and H probably due to the composition of end product as F used partially frozen meat with the addition of pork fat for sensory properties and not including skin in the product and included apart from chicken meat also turkey meat in the production of poultry meat preparations. Company H had only two growers as suppliers of live chickens, used no skin and the raw materials used for minced meat were 100% frozen.

Freezing is not the solution to the problem of Campylobacter contamination in chicken meat preparations, but is a contribution to reduction of levels of the pathogen. Our study shows that freezing products with skin attached to it, or, freezing meat and adding skin to it afterwards is limiting the effect of freezing on Campylobacter contamination level. The presence or adding skin leads to higher Campylobacter counts, and more positive samples. The reduction in Campylobacter concentration in frozen samples containing skin was not significant. Although all companies had a good documented and functional HACCP based system in place and achieved certification, a high variability for Campylobacter contamination between producers has been found. For this, there is a need to assess the status of current food safety management systems implemented in the various companies in order to know the constraints/bottlenecks for the variable performances of food safety management systems with

Fig. 5. Effect of the interaction between freezing and including of skin on the concentration of Campylobacter in Belgian chicken meat preparation (n = 656, 11 factories, February to December 2007). The dashed lines correspond to the 10 CFU/g (limit of quantification) and 100 CFU/g contamination levels.

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regard to the control of Campylobacter in these at risk products for campylobacteriosis. Acknowledgements This work was supported by a project fund from the Belgian Federal Public Services (FOD), Health, Food Chain Safety and Environment. We thank the eleven companies for their cooperation and commitment over the survey period. I. Habib is indebted to Ghent University (Belgium) for awarding the special research fund (BOF) fellowship. Ms. Josefien Gousseau is acknowledged for her professional technical assistance in performing Campylobacter analysis. References Anonymous, 2006a. ISO 10272 Microbiology of Food and Animal Feeding Stuffs — Horizontal Method for Detection and Enumeration of Campylobacter spp — Part 1: Detection Method. International Organisation for standardization (ISO), Geneva, Switzerland. Anonymous, 2006b. ISO 10272 Microbiology of Food and Animal Feeding Stuffs — Horizontal Method for Detection and Enumeration of Campylobacter spp — Part 2: Enumeration Method. International Organisation for standardization (ISO), Geneva, Switzerland. Anonymous, 2006c. Report of Task Force on Zoonoses Data Collection on Guidance Document on Good Practices for Design of Field Surveys. The EFSA Journal, vol. 93, pp. 1–24. Anonymous, 2006d. Report of Task Force on Zoonoses Data Collection on proposed technical specifications for a coordinated monitoring programme for Salmonella and Campylobacter in broiler meat in the EU. The EFSA Journal, vol. 92, pp. 1–33. Berrang, M.E., Ladely, S.R., Buhr, R.J., 2001. Presence and level of Campylobacter, Coliforms, Escherichia coli, and total aerobic bacteria recovered from broiler parts with and without skin. J. Food Prot. 64, 184–188 (5). Bhaduri, S., Cottrell, B., 2004. Survival of cold-stressed Campylobacter jejuni on ground chicken and chicken skin during frozen storage. Appl. Environ. Microbial. 70, 7103–7109. Chantarapanont, W., Berrang, M., Frank, J.F., 2003. Direct microscopic observation and viability determination of Campylobacter jejuni on chicken skin. J. Food Prot. 66, 2222–2230. Davis, M.A., Conner, D.E., 2007. Survival of Campylobacter jejuni on poultry skin and meat at varying temperatures. Poult. Sci. 86, 765–767. Friedman, C.R., Hoekstra, R.M., Samuel, M., Marcus, R., Bender, J., Shiferaw, B., Reddy, S., Ahuja, S.D., Helfrick, D.L., Hardnett, F., Carter, M., Anderson, B., Tauxe, R.V., Emerging Infections Program FoodNet Working Group, 2004. Risk factors for sporadic Campylobacter infection in the United States: a case–control study in FoodNet Sites. Clin. Infec. Dis. 38 (3), S285–296. Georgsson, F., Thornorkelsson, A.E., Geirsdóttir, M., Reiersen, J., Stern, N.J., 2006. The influence of freezing and duration of storage on Campylobacter and indicator bacteria in broiler carcasses. Food Microbiol. 23, 677–683. Ghafir, Y., China, B., Dierick, K., De Zutter, L., Daube, G., 2007. A seven-year survey of Campylobacter contamination in meat at different production stages in Belgium. Int. J. Food Microbiol. 116, 111–120. Gormley, F.J., Macrae, M., Forbes, K.J., Ogden, I.D., Dallas, J.F., Strachan, N.J., 2007. Has retail chicken played a role in the decline of human campylobacteriosis? Appl. Environ. Microbiol. doi:10.1128/AEM.01455-07. Habib, I., Sampers, I., Uyttendaele, M., Berkvens, D., De Zutter, L., 2008. Baseline data from a Belgium-wide survey of Campylobacter species contamination in chicken

303

meat preparations and considerations for a reliable monitoring program. Appl. Environ. Microbiol. 74, 5483–5489. Harrington, C.S., Mordhorst, H., Gravesen, A., Knøchel, S., 2007. Survival Characteristics of Campylobacter jejuni Strains under Different Food Processing Stresses at Chill Temperatures. Zoonoses and Public Health, vol. 54. Congress Campylobacter, Helicobacter & related organisms, Rotterdam, the Netherlands, pp. 128–129. s1. Humphrey, T., O'Brien, S., Madsen, M., 2007. Campylobacters as zoonotic pathogens: a food production perspective. Int. J. Food Microbiol. 117, 237–257. International Commission for Microbiological Specification in Foods, 2002. Process control. Microorganisms in Foods 7: Microbiological testing in food safety management. Kluwer Academic/Plenum Publishers, pp. 237–262. Jasson, V., Uyttendaele, M., Rajkovic, A., Debevere, J., 2007. Establishment of procedures provoking sub-lethal injury of Listeria monocytogenes, Campylobacter jejuni and Escherichia coli O157 to serve method performance testing. Int. J. Food. Microbial. 118, 241–249. Kärenlampi, R., Rautelin, H., Hänninen, M.L., 2007. Evaluation of genetic markers and molecular typing methods for prediction of sources of Campylobacter jejuni and C. coli infections. Appl. Environ. Microbial. 73 (5), 1683–1685. Lee, A., Smith, S.C., Coloe, P.J., 1998. Survival and growth of Campylobacter jejuni after artificial inoculation onto chicken skin as a function of temperature and packaging conditions. J. Food Prot. 61, 1609–1614 (6). Merchant, S., Blackall, P., Templeton, J., Price, E., Huygens, F., Giffard, P., 2007. Combinatorial Genotyping of Campylobacter jejuni: an attempt to identify the source of infection. Oral presentation 005. Proceedings of 14th International Workshop on Campylobacter, Helicobacter and Related Organisms (CHRO), 2–5 September, Rotterdam, Netherlands. Zoonoses and Public Health, 54, p. 2. Moore, J.E., Corcoran, D., Dooley, J.S., Fanning, S., Lucey, B., Matsuda, M., McDowell, D.A., Megraud, F., Millar, B.C., O'Mahony, R., O'Riordan, L., O'Rourke, M., Rao, J.R., Rooney, P.J., Sails, A., Whyte, P., 2005.. Campylobacter. Vet. Res. 36, 351–382. Ritz, M., Nauta, M.J., Teunis, P.F., van Leusden, F., Federighi, M., Havelaar, A.H., 2007. Modelling of Campylobacter survival in frozen chicken meat. J. Appl. Microbiol. 103 , 594–600. Scientific Institute of Public Health, Department Epidemiology, 2007. Surveillance van Infectieuze Aandoeningen door een Netwerk van Laboratoria voor Microbiologie 2006, Epidemiologisch Trends 1983–2005. Rapport: D/2007/2505/20. Solow, B.T., Cloak, O.M., Fratamico, P.M., 2003. Effect of temperature on viability of Campylobacter jejuni and Campylobacter coli on raw chicken or pork skin. J. Food Prot. 66, 2023–2031. StataCorp., 2007. Stata Statistical Software: Release 10. College Station, TX: StataCorp LP. Stern, N.J., Hiett, K.L., Alfredsson, G.A., Kristinsson, K.G., Reiersen, J., Hardardottir, H., Briem, H., Gunnarsson, E., Georgsson, F., Lowman, R., Berndtson, E., Lammerding, A. M., Paoli, G.M., Musgrove, M.T., 2003. Campylobacter spp. in Icelandic poultry operations and human disease. Epidemiol. Infect. 130, 23–32. Tam, C.C., Higgins, C.D., Rodrigues, L.C., Owen, R.J., Richardson, J.F., Curnow, J., Lamden, K., Millership, S., Neal, K., Patel, B., Sheridan, P., Wren, B.W., Al-Jaberi, S., McCarthy, N., O'Brien, S.J., 2007. Risk factors for reported Campylobacter enteritis in England: a case–control study. Oral presentation 006. Proceedings of 14th International Workshop on Campylobacter, Helicobacter and Related Organisms (CHRO), 2–5 September, Rotterdam, Netherlands. Zoonoses and Public Health, 54, pp. 2–3. Uyttendaele, M., Baert, K., Ghafir, Y., Daube, G., De Zutter, L., Herman, L., Dierick, K., Pierard, D., Dubois, J.J., Horion, B., Debevere, J., 2006. Quantitative risk assessment of Campylobacter spp. in poultry based meat preparations as one of the factors to support the development of risk-based microbiological criteria in Belgium. Int. J. Food Microbiol. 111, 149–163. Vandamme, P., Van Doorn, L.J., al Rashid, S.T., Quint, W.G., van der Plas, J., Chan, V.L., On, S.L., 1997. Campylobacter hyoilei Alderton et al., 1995 and Campylobacter coli Veron and Chatelain 1973 are subjective synonyms. Int. J. Syst. Bacteriol. 47, 1055–1060. Vellinga, A., Van Loock, F., 2002. The dioxin crisis as experiment to determine poultryrelated Campylobacter enteritis. J. Infec. Dis. 8, 19–22.