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Evidence of broiler meat contamination with post-disinfection strains of Campylobacter jejuni from slaughterhouse
International Journal of Food Microbiology 145 (2011) S116–S120
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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 ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Evidence of broiler meat contamination with post-disinfection strains of Campylobacter jejuni from slaughterhouse Eglė Kudirkienė a,⁎, Jurgita Bunevičienė a, Lone Brøndsted b, Hanne Ingmer b, John Elmerdahl Olsen b, Mindaugas Malakauskas a a b
Lithuanian Veterinary Academy, Department of Food Safety and Animal Hygiene, Tilžės 18, 47181, Kaunas, Lithuania University of Copenhagen, Department of Veterinary Disease Biology, Stigbøjlen 4, 1870 Frederiksberg C, Denmark
a r t i c l e
i n f o
Article history: Received 6 April 2010 Received in revised form 18 June 2010 Accepted 27 June 2010 Keywords: Campylobacter jejuni PCR-RFLP Disinfection Slaughterhouse Broilers
a b s t r a c t While cross-contamination from equipment and scalding water containing Campylobacter jejuni is considered the main route of broiler carcass contamination during slaughtering, alternative sources of C. jejuni may have been overlooked because only a limited number of studies focus on sampling of one broiler flock along the entire food chain and not many include the slaughterhouse environment. In the present study we have traced the changes of C. jejuni genotypes within one broiler flock from the beginning of rearing to the final product at the slaughterhouse with the aim to evaluate the dynamics and possible sources of carcass contamination with C. jejuni. Genotyping of 345 isolates of C. jejuni by flaA-RFLP revealed ten different flaA genotypes of C. jejuni along the broiler meat production chain. Broiler fillets were mainly contaminated with flaA genotypes found on the surfaces of slaughterhouse equipment and in the scalding water after cleaning and disinfection. Finally, it was clearly demonstrated that C. jejuni isolates remaining in the slaughterhouse environment after disinfection is a potential source of broiler meat contamination. Thus, identification of the mechanisms that allow such strains to persist in the slaughterhouse and survive cleaning is important for the establishment of future practices that will ensure sufficient reduction of C. jejuni in the slaughterhouse environment. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Campylobacter jejuni is a major food safety problem in the European countries as well as in the developing world. In 2008, a total of 190,566 confirmed human cases of campylobacteriosis was reported from 27 European Union countries, corresponding to an incidence of 40.7 per 100 000 of the population in EU (Anonymous, 2010). Despite numerous investigations and programs established to control Campylobacter during broiler processing, poultry meat remains the major source of human campylobacteriosis (Tam et al., 2009; Uyttendaele et al., 2006; Wingstrand et al., 2006). C. jejuni colonizes the intestine of poultry to very high numbers (Reich et al., 2008) and is the main source of poultry meat contamination during the slaughter process. The evisceration step is particularly critical, as intestinal contents of Campylobacter positive broilers may contaminate the meat especially if intestines are damaged (Allen et al., 2007). In addition, poultry meat from broiler flocks otherwise negative for Campylobacter may be contaminated if the previously slaughtered flock was positive and the bacteria remain on the surfaces of equipment in the slaughterhouse (Allen et al., 2007; ⁎ Corresponding author. Lithuanian Veterinary Academy, Tilžės g. 18, LT-43181, Kaunas, Lithuania. E-mail address: [email protected] (E. Kudirkienė). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.06.024
Alter et al., 2005) or in transport crates used for broiler transport from farm to slaughterhouse despite cleaning and disinfection (Hansson et al., 2005; Newell et al., 2001). Campylobacter cannot grow and survive well in the environment for longer periods (Alter and Scherer, 2006; Humphrey et al., 2007) and is sensitive to the disinfectants most commonly used in broiler meat industry (Peyrat et al., 2008a). Compared to other enteric organisms like Salmonella or E. coli, the survival mechanism of Campylobacter spp. is less known and many key regulators of the stress defense systems found in other organisms are not present in C. jejuni (Murphy et al., 2006). However, the ability to form biofilm on different surfaces and the ability to enter a viable but nonculturable state (VBNC) are putative mechanisms that may protect C. jejuni and allow its survival under unfavorable conditions in the food chain (Humphrey et al., 2007; Murphy et al., 2006; Park, 2002). Consequently, environments with adverse environmental conditions, such as the slaughterhouse, may select variants with a high ability to adapt to such conditions (Alter and Scherer, 2006; Klein et al., 2007; Takahashi et al., 2006). Recently, Johnsen et al. (2006) and Peyrat et al. (2008b) demonstrated that certain Campylobacter isolates are able to survive in the slaughterhouse environment over night and furthermore Peyrat et al. (2008b) demonstrated that they play a role in broiler carcass contamination during slaughter. However, it is necessary to further investigate the importance of such strains as a source of broiler
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meat contamination with C. jejuni and to determine the risk they possess to consumer health. In the current investigation, we have performed detailed sampling of the environment and broilers of selected broiler flock from farm to the end of slaughtering with the aim to understand the circulation of C. jejuni genotypes along the broiler meat production chain, to provide a clear understanding on the sources of broiler meat contamination, and to gain insight into the dynamics of different genotypes under adverse conditions at slaughterhouse. The flaA-RFLP method was used to determine genotypes due to its high discriminatory power similar to PFGE (Harrington et al., 2003) as well as accessibility and ease of use. The method is considered as stable for genotyping of Campylobacter spp. (de Boer et al., 2000) and has previously been used to investigate sources of poultry meat contamination with C. jejuni at slaughterhouses (Newell et al., 2001, Rivoal et al., 1999; Takahashi et al., 2006). 2. Materials and methods 2.1. Sample collection Two broiler flocks from the same farm were investigated. Environmental samples were taken inside the broiler houses prior to chicken placement, at delivery (delivery tray liners) and at 10 day intervals before slaughter and finally during slaughtering of the flock. During each visit at the farm (on days 15, 24 and 35) thirty broiler cloacae samples were collected according to a systematic walking pattern from five different points in the house. In total 232 samples were collected at farm level, 184 from broiler cloacae and 48 from the environment at farm and broiler houses, respectively. Campylobacter spp. positive samples were found in one flock. This was further sampled throughout the processing at the slaughterhouse. Environmental samples were taken at the slaughterhouse just before slaughtering, at the beginning of slaughter (first 3 h), when the slaughter process has been operating for 4–6 h and finally at the end of the day (7–8 h) (Table 1). Broiler cloacae, neck skin and breast fillet samples were collected at different stages during the slaughter
Table 1 Samples and isolates collected before and during processing of Campylobacter positive flock at slaughterhouse. Sample
Slaughtering period 0–3 ha
Environmental samples Tap water Air sample — dirty zone Floor of tractor used for delivering Floor — clean zone Floor — dirty zone Transporter Cutting machine Scalding water Plucking fingers Evisceration machine Conveyer belts (metal) — dirty zone Conveyer belts (plastic) — clean zone Carcass samples Broiler cloacae before scalding Neck skin after dethearing Neck skin after evisceration Neck skin after chilling Breast fillet after chilling Total a
0/1b 0/1 1/1 0/2 1/1 1/1 1/1 2/2 2/2 2/2 0/1 10/10 0/3 0/3 0/3 3/3 23/37
5c 5 4 1 5 10 8
49
15 102
4–6 h
7–8 h
NSd
NS 1/1e 0/1 0/1 0/1 NS NS 1/1 NS 0/1 NS 1/1
0/1 0/1 0/1 NS NS 1/1 0/1 0/1 1/1 NS 10/10 0/3 0/3 1/3 3/3 16/29
4
5
47
2 12 70
10/10 0/4 0/4 0/4 4/4 17/33
process including defeathering, evisceration and chilling (for details see Table 2). Samples of the processing environment at the slaughterhouse were also taken three times: (i) before broiler slaughtering (after disinfection), (ii) when the slaughter process has been operating for 4–6 h and (iii) at the end of the day (7–8 h). In total 100 samples were collected in the slaughterhouse (30 broiler cloacae samples, 30 neck skin samples, 10 breast fillets samples and 30 environmental samples). 2.2. Isolation of Campylobacter spp. Broiler cloacae samples collected at the farm and slaughterhouse were cultured by both direct plating and following selective enrichment while samples from tray liners of 1 day old chickens, water, environmental, neck skin and broiler fillets were cultured following selective enrichment alone. For direct plating, broiler cloacae samples taken with sterile cotton swabs moistened with saline (0.9% NaCl) were streaked on Campylobacter Blood-Free Selective Agar Base (modified CCDA-Preston) (CM0739; Oxoid Ltd., England) with CCDA Selective Supplement (SR0155E; Oxoid Ltd., England) plates that subsequently were incubated in a microaerophilic atmosphere (85% nitrogen, 10% carbon dioxide and 5% oxygen) generated by Campygen (CN25; Oxoid Ltd., England) at 37°C for 48 h. For selective enrichment, swabs were placed in a tube containing a Bolton selective enrichment broth (CM0983; Oxoid Ltd., England) with Bolton broth selective supplement (SR0183E; Oxoid Ltd., England) and 5% Laked horse blood (SR0048; OxoidLtd., England). Enrichment tubes were incubated microaerobically at 42 °C for 24– 42 h. Following enrichment, in the case when Campylobacter was not detected after direct plating, 10 μl of broth was streaked onto plates with mCCDA agar for Campylobacter spp. isolation. Air samples at the slaughterhouse were taken using an open mCCDA plate during all slaughtering processes. Plates were placed on racks 2 m above the floor. Tray liners were cut under sterile conditions Table 2 Distribution of Campylobacter jejuni isolates collected at farm and slaughterhouse according to results of genotyping by PCR-RFLP of the flaA gene. Origin of isolation
At farm Broiler cloacae samples 35 days Floor in anteroom
3
5
5 49
17 79
Environmental samples were taken before slaughtering process, while broiler samples randomly during first three hours of the processing. b Number of positive samples/number of samples collected. c Number of Campylobacter isolates. d Not sampled. e The plate for air sample was kept open from the beginning to end of slaughtering (0 to 8 h).
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At slaughterhouse 0-3 hoursa Broiler cloacae samples Floor (clean part) Transporter Cutting machine Evisceration machine Conveyer belts (dirty part) Scalding water Plucking fingers Breast fillet 4–6 h Broiler cloacae samples Neck skin after chilling Conveyer belts (dirty part) Scalding water Breast fillet 7–8 h Broiler cloacae samples Air sample Conveyer belts (clean part) Scalding water Breast fillet Total
flaA genotypes I
II
III
IV
V
-
-
-
-
-
13
2
-
-
-
10
2 -
-
-
-
24
3 7
4 4
2 2
1 1
1
VI
VII
VIII
IX
X
93 1
-
-
-
-
49
-
2 -
-
-
-
-
-
-
49 3 5 5 1 262
1 1
2
-
47 5 4
4 3 4 10 8 1 5 -
2
-
4 41
1 1
a At this time point the samples from the slaughterhouse environment were taken after disinfection/before slaughtering.
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and each of ½ parts were placed separately in two stomacher bags with 90 ml of Bolton broth. Environmental samples at the farm and slaughterhouse were taken using 10 × 10 gauze swab moistened with saline (0.9% NaCl) that was placed in a stomacher bag containing 90 ml of Bolton enrichment broth. The detection of Campylobacter in drinking water at the farm was performed by taking 500 ml of water from the water line in sterile bottles. The water was filtered using 0.45 μm pore size filter discs GN-6 Metricel®Grid (PALL Life sciences, Mexico). Filter discs were enriched microaerobically in 20 ml Bolton enrichment broth. Scalding water was scooped out from the water bath and 1 ml of it was added to 9 ml of Bolton broth for selective enrichment. Ten grams of neck skin and half of broiler fillets were placed into stomacher bags containing 90 ml of Bolton broth. All samples were further cultured as described above. Following isolation on mCCDA, up to five Campylobacter like colonies from each plate were picked, cells were examined by phasecontrast microscopy, and purified on blood agar plates (Blood Agar Base No. 2 (Liolfilchem, Italy) supplemented with 7% Laked horse blood and incubated at 37 °C for 1–2 days in microaerophilic atmosphere. All campylobacter isolates were subsequently stored at −80 °C in freezing broth (Nutrient broth No. 2 (CM67, Oxoid Ltd., England), Agar No. 1 (Liolfilchem, Italy) with 10% DMSO supplement (Merck, Germany) until further use. 2.3. Genomic DNA isolation Genomic DNA was extracted from 48 hour old cultures using GenEluteTM Bacterial Genomic DNA Kit (Sigma-Aldrich, Taufkirchen, Germany) according to the manufacture's instruction and stored at −20°C until further analysis. 2.4. Detection and differentiation of thermophilic Campylobacter spp. by multiplex PCR Campylobacter spp. isolates were identified at the species level by a slight modification of the method and primers described by Wang et al. (2002). Primers 23SF and 23SR created a 650 bp fragment which occurs in all Campylobacter spp. A 323-bp amplicon was generated for C. jejuni and a 126-bp amplicon was generated for C. coli by using a mixture of oligonucleotide primers hybridizing to the C. jejuni hipO gene (primers CJF and CJR) or the C. coli glyA gene (primers CCF and CCR). Each PCR mixture contained 2.0 μl of a 2 mM deoxynucleoside triphosphate mixture, 2.5 μl of 10× reaction buffer, 2.5 μl of 25 mM MgCl2, 0.2 μl of FastStart Taq DNA polymerase (Roche Diagnostics, GmbH, Mannheim, Germany), 0.75 μl of a 100 μM primer mixture containing 23S rRNA, Campylobacter jejuni and Campylobacter coli primers, 2.5 μl of chromosomal DNA, and MiliQ water to a final volume of 25 μl. The strain CNET019 was used as positive control for C. coli and the strain CNET002 as positive control for C. jejuni (Harrington et al. 2003). A negative control reaction containing ultrapure water instead of DNA template was also prepared. PCR products were analyzed by gel electrophoresis: 11.0 μl volume of each PCR product was loaded onto a 1.3% Top Vision LE GQ Agarose gel (MBI Fermentas, Vilnius, Lithuania) containing 0.05 μl/ml of ethidium bromide solution. The gel was visualized on an UV board. The GeneRulerTM100 bp DNA Ladder (MBI, Fermentas, Vilnius, Lithuania) was used as the molecular size marker. 2.5. flaA-RFLP typing A total of 345 isolates of C. jejuni from the farm (94) and the slaughterhouse (251) were genotyped. PCR-RFLP of the flaA gene was performed according to CAMPYNET method described previously (Harrington et al., 2003). Primers A1 5′-GGA TTT CGT ATT AAC ACA
AAT GGT GC and A2 5′-CTG TAG TAA TCT TAA AAC ATT TTG were used to amplify flaA gene of Campylobacter jejuni strains. The HpyF31 (DdeI) (MBI Fermentas, Vilnius, Lithuania) restriction enzyme was used for the restriction fragment length polymorphism (RFLP) analysis of the PCR product. flaA-RFLP gels were visualized on UV board and photographed. Images in TIFF format were imported into GelCompar II (Applied Maths, Kortrijk, Belgium). After pattern normalization, the similarity matrix was calculated using the Dice similarity coefficient and clustering by the Unweighted Paired Group Method with Arithmetic mean values (UPGMA). Band position tolerance and the optimization coefficient were set to 2.0%. The GeneRulerTM100 bp plus DNA Ladder (MBI, Fermentas, Lithuania) was used as the molecular size marker. 3. Results 3.1. Occurrence of Campylobacter spp. in broiler cloacae and farm environment In August and September of 2008 we followed the C. jejuni colonization of broilers at a farm which, according to a previous investigation (Kudirkienė et al., 2010), could be expected to have a high prevalence of Campylobacter spp. during this period of year. Two flocks were examined from the beginning until the end of rearing. One of the two flocks was positive for Campyloacter spp. Bacteria were not detected in the farm environment nor in broiler cloacae samples on days 1, 15 and 24 of rearing. However, on days 35, 28 out of 30 collected cloacal samples (93.3%) were positive for C. jejuni. Additionally, one out of 48 environmental samples was positive for C. jejuni on the 35th day of sampling. 3.2. Occurrence of Campylobacter spp. in broiler cloacae, carcasses and slaughterhouse environment The positive flock was also followed during slaughtering. We sampled the slaughter house environment before slaughter, but after disinfection, and found 10 of 15 samples (66.6%) positive for C. jejuni (Table 1). In two of those samples C. coli was also identified. After 4–6 h of slaughtering, 2 of the 7 environmental samples were positive for C. jejuni and at the end of the day (7–8 h after the beginning of slaughter) C. jejuni was identified in 2 environmental samples out of 6 collected. The air sample from the clean zone was negative while the ones obtained from the dirty zone during all slaughtering were positive for C. jejuni. Broilers were also sampled after killing when hanging on the line. C. jejuni was isolated in all out of 30 broiler cloacae samples collected. All 10 broiler fillets samples collected at the end of the slaughter line were positive for C. jejuni as well. Meanwhile, C. jejuni was detected only in one neck skin sample out of all collected. 3.3. flaA-RFLP genotyping of C. jejuni isolates The genotypic characterization of 345 strains of C. jejuni revealed ten (named from I to X, respectively) different flaA genotypes when a cut-off value of 100% similarity was used (Fig. 1). Only one genotype (VI) was identified among the C. jejuni isolated from the 148 broiler cloacae samples taken both at the farm and prior to slaughter at the slaughterhouse (Table 2). Moreover, the same genotype was also detected in the scalding water and on conveyer belt samples after 4–6 h of slaughter and at the end of the day. Interestingly, this C. jejuni genotype VI was found only on one breast fillet sample. In addition, this genotype also appeared in the air sample. Three flaA genotypes of C. jejuni (I, VIII and IX respectively) were found in the slaughterhouse environment after disinfection when samples were taken 1 h before slaughter. These genotypes were not present in environmental samples taken during broiler processing,
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Fig. 1. PCR-RFLP profiles of flaA gene cut with HpyF31 (DdeI) from strains of C. jejuni representing ten flaA genotypes determined in this study.
however genotypes I and IX dominated on breast fillet samples at the end of the day. Genotype I was detected in 23 (52.3%) and genotype IX in 6 (13.6%) out of 44 C. jejuni isolates originating from the breast fillets. Only one sample obtained from the neck skin was positive for Campylobacter, however the same flaA genotype (II) was found on broiler fillets as well. Additional genotypes (III, IV, VII, V and X) were isolated from broiler fillets, however these genotypes were not detected either in cloacae samples taken at the farm or in the environment during slaughtering. 4. Discussion The aim of the present study was to track the changes of C. jejuni genotypes within a Campylobacter positive broiler flock along the poultry meat production chain in order to gain insight into the dynamics of broiler meat contamination. We found only one genotype (VI) of C. jejuni in broiler cloacae at the farm and when broilers were sampled at the slaughterhouse just before slaughtering. Usually the presence of more than one genotype within broiler flocks is reported including our previous studies at the same poultry farm (Berndtson et al. 1996; Hiett et al. 2002; Kudirkienė et al., 2010). However, in most studies one or two genotypes dominate in a flock while the remaining genotypes only are found sporadically. The presence of only one clone of C. jejuni in the present study may be explained by the ability of some clones to displace others in the chicken intestinal tract and colonize chickens consistently, as previously shown in the study of Calderon-Gomez et al. (2008). Besides, we only isolated Campylobacter from broiler cloacae by direct plating and additional genotypes may not be detected without an enrichment step if they were present in low numbers. The genotype detected during broiler rearing at the farm was also isolated in the slaughterhouse environment (surfaces of equipment, scalding water, and air), in agreement with other studies (Klein et al., 2007; Lienau et al., 2007; Newell et al., 2001) showing that Campylobacter is introduced into the slaughterhouse by Campylobacter positive broiler flocks. However, even though genotype VI dominated in the broiler cloacae and environmental samples at the slaughterhouse during processing, this particular genotype was only detected on one breast fillet sample at the end of the chain. It may be speculated that C. jejuni isolates belonging to this genotype could not survive the conditions during slaughtering such as low chilling temperature or high temperature of scalding water (Alter and Scherer, 2006; Humphrey et al., 2007; Murphy et al., 2006) and only a few cells survived until the end of processing. In the present study the dominant genotypes (I and IX) detected on broiler fillets were those also found in the slaughterhouse
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environment after disinfection before slaughter. Out of three different genotypes (I, VIII and IX) identified among isolates collected before slaughtering, genotype I together with genotype IX became dominant on broiler fillets. This is in sharp contrast to previous studies (Lienau et al., 2007; Takahashi et al. 2006), which showed that the final products were mainly contaminated with the genotypes carried by the broilers at the farm as well as genotypes isolated from slaughterhouse environment during processing and transport crates after cleaning and disinfection. It should however be noted that these studies did not include sampling of the environment of the slaughterhouse after disinfection and thus could not assess the importance of environmental contamination. It could be speculated that persistent isolates may have particular traits that allow survival in the slaughterhouse environment however; this needs to be confirmed by further characterization of the strains. To our knowledge only two additional studies have sampled and isolated C. jejuni in the environment of the slaughterhouse after disinfection (Johnsen et al. 2006; Peyrat et al. (2008a,b). Peyrat et al. (2008b) demonstrated that C. jejuni genotypes found after disinfection may contaminate broiler neck skin during slaughtering, however contamination with same genotypes from transport crates was not excluded in the study. Presently we cannot conclude whether the C. jejuni genotypes I and IX detected in our study had persisted in the slaughterhouse environment for a longer time period or if it just remained for a short while due to insufficient cleaning and disinfection procedures. Residues of organic matter, feathers on rubber fingers of the defeathering machine and scalding water were visually detected during sampling after disinfection, and these might increase the ability of C. jejuni to survive during disinfection procedure. In addition, failure of disinfection including too low concentration or too short application time of the disinfectants is possible. Interestingly, we were able to isolate C. jejuni from scalding water not only during processing, but also before the slaughtering, using an enrichment procedure for the isolation of Campylobacter spp. The presence of C. jejuni in scalding water has not been found in previous studies (Allen et al., 2007; Newell et al., 2001; Peyrat et al., 2008a), most likely because the isolation procedure used did not allow the detection of low numbers or sub-lethal damaged bacteria. Most strains of C. jejuni are susceptible to chlorine compounds (Blaser et al., 1986) and quaternary ammonium compounds (Peyrat et al., 2008a), the most commonly used disinfectant in broiler slaughterhouses. However, such strains may be protected from biocidal activities if they form a biofilm (Buswell et al., 1998) and the ability of other food borne pathogens like Listeria monocytogenes to survive in the slaughterhouse due to biofilm formation is well described (Gandhi and Chikindas, 2007). Recent studies carried out by Klein et al. (2007) and Takahashi et al. (2006) showed that even though C. jejuni isolates from the farm were highly diverse, only those genotypes that were most tolerant to environmental stresses were able to survive the slaughtering process and contaminate the final product (Klein et al., 2007). In agreement with this, the number of genotypes decreased notably from the beginning until the end of the slaughter line (Hiett et al., 2002; Klein et al., 2007). In contrast to this and other reports (Lindmark et al., 2006; Newell et al., 2001) we found several different C. jejuni genotypes on the final product and an increased diversity during the day. Only three different genotypes (I, II, and IX) were detected at the beginning of slaughter and after 4–6 h, while eight different genotypes (IX, III, II, IV, VII, V, VI, and X) could be found at the end of the day. Several factors may influence the results. First of all, the enrichment procedure was only used for environmental and not cloacae samples, which may bias the result as differences in genotypes detected using enrichment and direct plating were demonstrated by Newell et al. (2001). Secondly, the change of flaA genotype cannot be completely excluded as the reason of the rise of new genotypes on the breast fillets (de Boer et al., 2000). The
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intragenomic recombination between flaA and flaB genes between two strains resulting in appearance or disappearance of the new bands was demonstrated by Harrington et al. (1997). Against, the latter theory speaks that the conditions at the slaughterhouse are not favorable for growth of C. jejuni, therefore new genotypic variants do not occur frequently in the slaughterhouse. However, testing of genotypes found at the end of processing with another genotyping method(s) would be helpful to get optimal answer how the strains differ. 5. Conclusions The present study provides evidence that C. jejuni isolates present in the slaughterhouse environment after disinfection are an important source of broiler meat contamination. In contrast to previous studies, the genotypes of C. jejuni found on the surfaces of the equipment and in the scalding water of slaughterhouse after disinfection were also the dominating genotypes on broiler fillets, whereas contamination of broiler fillets with the C. jejuni genotype colonizing broilers during the rearing period was not significant in our study. Further characterization of the C. jejuni strains obtained along the entire meat production chain will be useful for our understanding of the survival mechanisms of C. jejuni in the adverse environment of the slaughterhouse, thus will allow us to improve the practices used to control C. jejuni in slaughterhouses. Acknowledgments This study was financially supported by the European Union funded Integrated Project BIOTRACER (contract FOOD-2006-CT036272) under the 6th RTD Framework. References Allen, V.M., Bull, S.A., Corry, J.E., Domingue, G., Jorgensen, F., Frost, J.A., Whyte, R., Gonzalez, A., Elviss, N., Humphrey, T.J., 2007. Campylobacter spp. contamination of chicken carcasses during processing in relation to flock colonisation. International Journal of Food Microbiology 113, 54–61. Alter, T., Scherer, K., 2006. Stress response of Campylobacter spp. and its role in food processing. Journal of Veterinary Medicine 53, 351–357. Alter, T., Gaull, F., Froeb, A., Fehlhaber, K., 2005. Distribution of Campylobacter jejuni strains at different stages of a turkey slaughter line. Food Microbiology 22, 345–351. Anonymous, 2010. The Community Summary Report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in the European Union in 2008. The EFSA Journal 1496, 124–127. Berndtson, E., Emanuelson, U., Engvall, A., Danielsson-Tham, M.L., 1996. A 1-year epidemiological study of campylobacters in 18 Swedish farms. Preventive Veterinary Medicine 26, 167–185. Blaser, M.J., Smith, P.F., Wang, W.L., Hoff, J.C., 1986. Inactivation of Campylobacter jejuni by chlorine and monochloramine. Applied and Environmental Microbiology 51, 307–311. Buswell, C.M., Herlihy, Y.M., Lawrence, L.M., McGuiggan, J.T., Marsh, P.D., Keevil, C.W., Leach, S.A., 1998. Extended survival and persistence of Campylobacter spp. in water and aquatic biofilms and their detection by immunofluorescent-antibody and rRNA staining. Applied and Environmental Microbiology 64, 733–741. Calderon-Gomez, L.I., Hartley, L.E., McCormack, A., Ringoir, D.D., Korolik, V., 2008. Potential use of characterised hyper-colonising strain(s) of Campylobacter jejuni to reduce circulation of environmental strains in commercial poultry. Veterinary Microbiology 134, 353–361. De Boer, P., Duim, B., Rigter, A., Van der Plas, J., Jacobs-Reitsma, W.F., Wagenaar, J.A., 2000. Computer-assisted analysis and epidemiological value of genotyping methods for Campylobacter jejuni and Campylobacter coli. Journal of Clinical Microbiology 38, 1940–1946. Gandhi, M., Chikindas, M.L., 2007. Listeria: a foodborne pathogen that knows how to survive. International Journal of Food Microbiology 113, 1–15.
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Lindmark, H., Dietrich, C., Andersson, L., Lindqvist, R., Olsson Engvall, E., 2006. Distribution of Campylobacter genotypes on broilers during slaughter. Journal of Food Protection 69, 2902–2907. Murphy, C., Carroll, C., Jordan, K.N., 2006. Environmental survival mechanism of the foodborne pathogen Campylobacter jejuni. Journal of Applied Microbiology 100, 623–632. Newell, D.G., Shreeve, J.E., Toszeghy, M., Domingue, G., Bull, S., Humphrey, T., Mead, G., 2001. Changes in the carriage of Campylobacter strains by poultry carcasses during processing in abattoirs. Applied and Environmental Microbiology 67, 2636–2640. Park, S., 2002. The physiology of Campylobacter species and its relevance to their role as foodborne pathogens. International Journal of Food Microbiology 74, 177–188. Peyrat, M.B., Soumet, C., Maris, P., Sanders, P., 2008a. Phenotypes and genotypes of campylobacter strains isolated after cleaning and disinfection in poultry slaughterhouses. Veterinary Microbiology 128, 313–326. Peyrat, M.B., Soumet, C., Maris, P., Sanders, P., 2008b. Recovery of Campylobacter jejuni from surfaces of poultry slaughterhouses after cleaning and disinfection procedures: analysis of a potential source of carcass contamination. International Journal of Food Microbiology 124, 188–194. Reich, F., Atanassova, V., Haunhorst, E., Klein, G., 2008. The effect of Campylobacter numbers in ceaca on the contamination of broiler carcasses with Campylobacter. International Journal of Food Microbiology 127, 116–120. Rivoal, K., Denis, M., Salvat, G., Colin, P., Ermel, G., 1999. Molecular characterization of the diversity of Campylobacter spp. isolates collected from a poultry slaughterhouse: analysis of cross-contamination. Letters in Applied Microbiology 29, 370–374. Takahashi, R., Shahada, F., Chuma, T., Okamoto, K., 2006. Analysis of Campylobacter spp. contamination in broiler from the farm to the final meat cuts by using restriction fragment length polymorphism of the polymerase chain reaction products. International Journal of Food Microbiology 110, 240–245. Tam, C.C., Higgins, C.D., Neal, K.R., Rodrigues, L.C., Millership, S.E., O'Brien, S.J., 2009. Chicken consumption and use of acid-suppressing medications as risk factors for Campylobacter enteritis, England. Emerging Infectious Diseases 15, 1402–1408. 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. International Journal of Food Microbiology 111, 149–163. Wang, G., Clark, C.G., Taylor, T.M., Pucknell, C., Barton, C., Price, L., Woodward, D.L., Rodgers, F.G., 2002. 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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 ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Evidence of broiler meat contamination with post-disinfection strains of Campylobacter jejuni from slaughterhouse Eglė Kudirkienė a,⁎, Jurgita Bunevičienė a, Lone Brøndsted b, Hanne Ingmer b, John Elmerdahl Olsen b, Mindaugas Malakauskas a a b
Lithuanian Veterinary Academy, Department of Food Safety and Animal Hygiene, Tilžės 18, 47181, Kaunas, Lithuania University of Copenhagen, Department of Veterinary Disease Biology, Stigbøjlen 4, 1870 Frederiksberg C, Denmark
a r t i c l e
i n f o
Article history: Received 6 April 2010 Received in revised form 18 June 2010 Accepted 27 June 2010 Keywords: Campylobacter jejuni PCR-RFLP Disinfection Slaughterhouse Broilers
a b s t r a c t While cross-contamination from equipment and scalding water containing Campylobacter jejuni is considered the main route of broiler carcass contamination during slaughtering, alternative sources of C. jejuni may have been overlooked because only a limited number of studies focus on sampling of one broiler flock along the entire food chain and not many include the slaughterhouse environment. In the present study we have traced the changes of C. jejuni genotypes within one broiler flock from the beginning of rearing to the final product at the slaughterhouse with the aim to evaluate the dynamics and possible sources of carcass contamination with C. jejuni. Genotyping of 345 isolates of C. jejuni by flaA-RFLP revealed ten different flaA genotypes of C. jejuni along the broiler meat production chain. Broiler fillets were mainly contaminated with flaA genotypes found on the surfaces of slaughterhouse equipment and in the scalding water after cleaning and disinfection. Finally, it was clearly demonstrated that C. jejuni isolates remaining in the slaughterhouse environment after disinfection is a potential source of broiler meat contamination. Thus, identification of the mechanisms that allow such strains to persist in the slaughterhouse and survive cleaning is important for the establishment of future practices that will ensure sufficient reduction of C. jejuni in the slaughterhouse environment. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Campylobacter jejuni is a major food safety problem in the European countries as well as in the developing world. In 2008, a total of 190,566 confirmed human cases of campylobacteriosis was reported from 27 European Union countries, corresponding to an incidence of 40.7 per 100 000 of the population in EU (Anonymous, 2010). Despite numerous investigations and programs established to control Campylobacter during broiler processing, poultry meat remains the major source of human campylobacteriosis (Tam et al., 2009; Uyttendaele et al., 2006; Wingstrand et al., 2006). C. jejuni colonizes the intestine of poultry to very high numbers (Reich et al., 2008) and is the main source of poultry meat contamination during the slaughter process. The evisceration step is particularly critical, as intestinal contents of Campylobacter positive broilers may contaminate the meat especially if intestines are damaged (Allen et al., 2007). In addition, poultry meat from broiler flocks otherwise negative for Campylobacter may be contaminated if the previously slaughtered flock was positive and the bacteria remain on the surfaces of equipment in the slaughterhouse (Allen et al., 2007; ⁎ Corresponding author. Lithuanian Veterinary Academy, Tilžės g. 18, LT-43181, Kaunas, Lithuania. E-mail address: [email protected] (E. Kudirkienė). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.06.024
Alter et al., 2005) or in transport crates used for broiler transport from farm to slaughterhouse despite cleaning and disinfection (Hansson et al., 2005; Newell et al., 2001). Campylobacter cannot grow and survive well in the environment for longer periods (Alter and Scherer, 2006; Humphrey et al., 2007) and is sensitive to the disinfectants most commonly used in broiler meat industry (Peyrat et al., 2008a). Compared to other enteric organisms like Salmonella or E. coli, the survival mechanism of Campylobacter spp. is less known and many key regulators of the stress defense systems found in other organisms are not present in C. jejuni (Murphy et al., 2006). However, the ability to form biofilm on different surfaces and the ability to enter a viable but nonculturable state (VBNC) are putative mechanisms that may protect C. jejuni and allow its survival under unfavorable conditions in the food chain (Humphrey et al., 2007; Murphy et al., 2006; Park, 2002). Consequently, environments with adverse environmental conditions, such as the slaughterhouse, may select variants with a high ability to adapt to such conditions (Alter and Scherer, 2006; Klein et al., 2007; Takahashi et al., 2006). Recently, Johnsen et al. (2006) and Peyrat et al. (2008b) demonstrated that certain Campylobacter isolates are able to survive in the slaughterhouse environment over night and furthermore Peyrat et al. (2008b) demonstrated that they play a role in broiler carcass contamination during slaughter. However, it is necessary to further investigate the importance of such strains as a source of broiler
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meat contamination with C. jejuni and to determine the risk they possess to consumer health. In the current investigation, we have performed detailed sampling of the environment and broilers of selected broiler flock from farm to the end of slaughtering with the aim to understand the circulation of C. jejuni genotypes along the broiler meat production chain, to provide a clear understanding on the sources of broiler meat contamination, and to gain insight into the dynamics of different genotypes under adverse conditions at slaughterhouse. The flaA-RFLP method was used to determine genotypes due to its high discriminatory power similar to PFGE (Harrington et al., 2003) as well as accessibility and ease of use. The method is considered as stable for genotyping of Campylobacter spp. (de Boer et al., 2000) and has previously been used to investigate sources of poultry meat contamination with C. jejuni at slaughterhouses (Newell et al., 2001, Rivoal et al., 1999; Takahashi et al., 2006). 2. Materials and methods 2.1. Sample collection Two broiler flocks from the same farm were investigated. Environmental samples were taken inside the broiler houses prior to chicken placement, at delivery (delivery tray liners) and at 10 day intervals before slaughter and finally during slaughtering of the flock. During each visit at the farm (on days 15, 24 and 35) thirty broiler cloacae samples were collected according to a systematic walking pattern from five different points in the house. In total 232 samples were collected at farm level, 184 from broiler cloacae and 48 from the environment at farm and broiler houses, respectively. Campylobacter spp. positive samples were found in one flock. This was further sampled throughout the processing at the slaughterhouse. Environmental samples were taken at the slaughterhouse just before slaughtering, at the beginning of slaughter (first 3 h), when the slaughter process has been operating for 4–6 h and finally at the end of the day (7–8 h) (Table 1). Broiler cloacae, neck skin and breast fillet samples were collected at different stages during the slaughter
Table 1 Samples and isolates collected before and during processing of Campylobacter positive flock at slaughterhouse. Sample
Slaughtering period 0–3 ha
Environmental samples Tap water Air sample — dirty zone Floor of tractor used for delivering Floor — clean zone Floor — dirty zone Transporter Cutting machine Scalding water Plucking fingers Evisceration machine Conveyer belts (metal) — dirty zone Conveyer belts (plastic) — clean zone Carcass samples Broiler cloacae before scalding Neck skin after dethearing Neck skin after evisceration Neck skin after chilling Breast fillet after chilling Total a
0/1b 0/1 1/1 0/2 1/1 1/1 1/1 2/2 2/2 2/2 0/1 10/10 0/3 0/3 0/3 3/3 23/37
5c 5 4 1 5 10 8
49
15 102
4–6 h
7–8 h
NSd
NS 1/1e 0/1 0/1 0/1 NS NS 1/1 NS 0/1 NS 1/1
0/1 0/1 0/1 NS NS 1/1 0/1 0/1 1/1 NS 10/10 0/3 0/3 1/3 3/3 16/29
4
5
47
2 12 70
10/10 0/4 0/4 0/4 4/4 17/33
process including defeathering, evisceration and chilling (for details see Table 2). Samples of the processing environment at the slaughterhouse were also taken three times: (i) before broiler slaughtering (after disinfection), (ii) when the slaughter process has been operating for 4–6 h and (iii) at the end of the day (7–8 h). In total 100 samples were collected in the slaughterhouse (30 broiler cloacae samples, 30 neck skin samples, 10 breast fillets samples and 30 environmental samples). 2.2. Isolation of Campylobacter spp. Broiler cloacae samples collected at the farm and slaughterhouse were cultured by both direct plating and following selective enrichment while samples from tray liners of 1 day old chickens, water, environmental, neck skin and broiler fillets were cultured following selective enrichment alone. For direct plating, broiler cloacae samples taken with sterile cotton swabs moistened with saline (0.9% NaCl) were streaked on Campylobacter Blood-Free Selective Agar Base (modified CCDA-Preston) (CM0739; Oxoid Ltd., England) with CCDA Selective Supplement (SR0155E; Oxoid Ltd., England) plates that subsequently were incubated in a microaerophilic atmosphere (85% nitrogen, 10% carbon dioxide and 5% oxygen) generated by Campygen (CN25; Oxoid Ltd., England) at 37°C for 48 h. For selective enrichment, swabs were placed in a tube containing a Bolton selective enrichment broth (CM0983; Oxoid Ltd., England) with Bolton broth selective supplement (SR0183E; Oxoid Ltd., England) and 5% Laked horse blood (SR0048; OxoidLtd., England). Enrichment tubes were incubated microaerobically at 42 °C for 24– 42 h. Following enrichment, in the case when Campylobacter was not detected after direct plating, 10 μl of broth was streaked onto plates with mCCDA agar for Campylobacter spp. isolation. Air samples at the slaughterhouse were taken using an open mCCDA plate during all slaughtering processes. Plates were placed on racks 2 m above the floor. Tray liners were cut under sterile conditions Table 2 Distribution of Campylobacter jejuni isolates collected at farm and slaughterhouse according to results of genotyping by PCR-RFLP of the flaA gene. Origin of isolation
At farm Broiler cloacae samples 35 days Floor in anteroom
3
5
5 49
17 79
Environmental samples were taken before slaughtering process, while broiler samples randomly during first three hours of the processing. b Number of positive samples/number of samples collected. c Number of Campylobacter isolates. d Not sampled. e The plate for air sample was kept open from the beginning to end of slaughtering (0 to 8 h).
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At slaughterhouse 0-3 hoursa Broiler cloacae samples Floor (clean part) Transporter Cutting machine Evisceration machine Conveyer belts (dirty part) Scalding water Plucking fingers Breast fillet 4–6 h Broiler cloacae samples Neck skin after chilling Conveyer belts (dirty part) Scalding water Breast fillet 7–8 h Broiler cloacae samples Air sample Conveyer belts (clean part) Scalding water Breast fillet Total
flaA genotypes I
II
III
IV
V
-
-
-
-
-
13
2
-
-
-
10
2 -
-
-
-
24
3 7
4 4
2 2
1 1
1
VI
VII
VIII
IX
X
93 1
-
-
-
-
49
-
2 -
-
-
-
-
-
-
49 3 5 5 1 262
1 1
2
-
47 5 4
4 3 4 10 8 1 5 -
2
-
4 41
1 1
a At this time point the samples from the slaughterhouse environment were taken after disinfection/before slaughtering.
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and each of ½ parts were placed separately in two stomacher bags with 90 ml of Bolton broth. Environmental samples at the farm and slaughterhouse were taken using 10 × 10 gauze swab moistened with saline (0.9% NaCl) that was placed in a stomacher bag containing 90 ml of Bolton enrichment broth. The detection of Campylobacter in drinking water at the farm was performed by taking 500 ml of water from the water line in sterile bottles. The water was filtered using 0.45 μm pore size filter discs GN-6 Metricel®Grid (PALL Life sciences, Mexico). Filter discs were enriched microaerobically in 20 ml Bolton enrichment broth. Scalding water was scooped out from the water bath and 1 ml of it was added to 9 ml of Bolton broth for selective enrichment. Ten grams of neck skin and half of broiler fillets were placed into stomacher bags containing 90 ml of Bolton broth. All samples were further cultured as described above. Following isolation on mCCDA, up to five Campylobacter like colonies from each plate were picked, cells were examined by phasecontrast microscopy, and purified on blood agar plates (Blood Agar Base No. 2 (Liolfilchem, Italy) supplemented with 7% Laked horse blood and incubated at 37 °C for 1–2 days in microaerophilic atmosphere. All campylobacter isolates were subsequently stored at −80 °C in freezing broth (Nutrient broth No. 2 (CM67, Oxoid Ltd., England), Agar No. 1 (Liolfilchem, Italy) with 10% DMSO supplement (Merck, Germany) until further use. 2.3. Genomic DNA isolation Genomic DNA was extracted from 48 hour old cultures using GenEluteTM Bacterial Genomic DNA Kit (Sigma-Aldrich, Taufkirchen, Germany) according to the manufacture's instruction and stored at −20°C until further analysis. 2.4. Detection and differentiation of thermophilic Campylobacter spp. by multiplex PCR Campylobacter spp. isolates were identified at the species level by a slight modification of the method and primers described by Wang et al. (2002). Primers 23SF and 23SR created a 650 bp fragment which occurs in all Campylobacter spp. A 323-bp amplicon was generated for C. jejuni and a 126-bp amplicon was generated for C. coli by using a mixture of oligonucleotide primers hybridizing to the C. jejuni hipO gene (primers CJF and CJR) or the C. coli glyA gene (primers CCF and CCR). Each PCR mixture contained 2.0 μl of a 2 mM deoxynucleoside triphosphate mixture, 2.5 μl of 10× reaction buffer, 2.5 μl of 25 mM MgCl2, 0.2 μl of FastStart Taq DNA polymerase (Roche Diagnostics, GmbH, Mannheim, Germany), 0.75 μl of a 100 μM primer mixture containing 23S rRNA, Campylobacter jejuni and Campylobacter coli primers, 2.5 μl of chromosomal DNA, and MiliQ water to a final volume of 25 μl. The strain CNET019 was used as positive control for C. coli and the strain CNET002 as positive control for C. jejuni (Harrington et al. 2003). A negative control reaction containing ultrapure water instead of DNA template was also prepared. PCR products were analyzed by gel electrophoresis: 11.0 μl volume of each PCR product was loaded onto a 1.3% Top Vision LE GQ Agarose gel (MBI Fermentas, Vilnius, Lithuania) containing 0.05 μl/ml of ethidium bromide solution. The gel was visualized on an UV board. The GeneRulerTM100 bp DNA Ladder (MBI, Fermentas, Vilnius, Lithuania) was used as the molecular size marker. 2.5. flaA-RFLP typing A total of 345 isolates of C. jejuni from the farm (94) and the slaughterhouse (251) were genotyped. PCR-RFLP of the flaA gene was performed according to CAMPYNET method described previously (Harrington et al., 2003). Primers A1 5′-GGA TTT CGT ATT AAC ACA
AAT GGT GC and A2 5′-CTG TAG TAA TCT TAA AAC ATT TTG were used to amplify flaA gene of Campylobacter jejuni strains. The HpyF31 (DdeI) (MBI Fermentas, Vilnius, Lithuania) restriction enzyme was used for the restriction fragment length polymorphism (RFLP) analysis of the PCR product. flaA-RFLP gels were visualized on UV board and photographed. Images in TIFF format were imported into GelCompar II (Applied Maths, Kortrijk, Belgium). After pattern normalization, the similarity matrix was calculated using the Dice similarity coefficient and clustering by the Unweighted Paired Group Method with Arithmetic mean values (UPGMA). Band position tolerance and the optimization coefficient were set to 2.0%. The GeneRulerTM100 bp plus DNA Ladder (MBI, Fermentas, Lithuania) was used as the molecular size marker. 3. Results 3.1. Occurrence of Campylobacter spp. in broiler cloacae and farm environment In August and September of 2008 we followed the C. jejuni colonization of broilers at a farm which, according to a previous investigation (Kudirkienė et al., 2010), could be expected to have a high prevalence of Campylobacter spp. during this period of year. Two flocks were examined from the beginning until the end of rearing. One of the two flocks was positive for Campyloacter spp. Bacteria were not detected in the farm environment nor in broiler cloacae samples on days 1, 15 and 24 of rearing. However, on days 35, 28 out of 30 collected cloacal samples (93.3%) were positive for C. jejuni. Additionally, one out of 48 environmental samples was positive for C. jejuni on the 35th day of sampling. 3.2. Occurrence of Campylobacter spp. in broiler cloacae, carcasses and slaughterhouse environment The positive flock was also followed during slaughtering. We sampled the slaughter house environment before slaughter, but after disinfection, and found 10 of 15 samples (66.6%) positive for C. jejuni (Table 1). In two of those samples C. coli was also identified. After 4–6 h of slaughtering, 2 of the 7 environmental samples were positive for C. jejuni and at the end of the day (7–8 h after the beginning of slaughter) C. jejuni was identified in 2 environmental samples out of 6 collected. The air sample from the clean zone was negative while the ones obtained from the dirty zone during all slaughtering were positive for C. jejuni. Broilers were also sampled after killing when hanging on the line. C. jejuni was isolated in all out of 30 broiler cloacae samples collected. All 10 broiler fillets samples collected at the end of the slaughter line were positive for C. jejuni as well. Meanwhile, C. jejuni was detected only in one neck skin sample out of all collected. 3.3. flaA-RFLP genotyping of C. jejuni isolates The genotypic characterization of 345 strains of C. jejuni revealed ten (named from I to X, respectively) different flaA genotypes when a cut-off value of 100% similarity was used (Fig. 1). Only one genotype (VI) was identified among the C. jejuni isolated from the 148 broiler cloacae samples taken both at the farm and prior to slaughter at the slaughterhouse (Table 2). Moreover, the same genotype was also detected in the scalding water and on conveyer belt samples after 4–6 h of slaughter and at the end of the day. Interestingly, this C. jejuni genotype VI was found only on one breast fillet sample. In addition, this genotype also appeared in the air sample. Three flaA genotypes of C. jejuni (I, VIII and IX respectively) were found in the slaughterhouse environment after disinfection when samples were taken 1 h before slaughter. These genotypes were not present in environmental samples taken during broiler processing,
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Fig. 1. PCR-RFLP profiles of flaA gene cut with HpyF31 (DdeI) from strains of C. jejuni representing ten flaA genotypes determined in this study.
however genotypes I and IX dominated on breast fillet samples at the end of the day. Genotype I was detected in 23 (52.3%) and genotype IX in 6 (13.6%) out of 44 C. jejuni isolates originating from the breast fillets. Only one sample obtained from the neck skin was positive for Campylobacter, however the same flaA genotype (II) was found on broiler fillets as well. Additional genotypes (III, IV, VII, V and X) were isolated from broiler fillets, however these genotypes were not detected either in cloacae samples taken at the farm or in the environment during slaughtering. 4. Discussion The aim of the present study was to track the changes of C. jejuni genotypes within a Campylobacter positive broiler flock along the poultry meat production chain in order to gain insight into the dynamics of broiler meat contamination. We found only one genotype (VI) of C. jejuni in broiler cloacae at the farm and when broilers were sampled at the slaughterhouse just before slaughtering. Usually the presence of more than one genotype within broiler flocks is reported including our previous studies at the same poultry farm (Berndtson et al. 1996; Hiett et al. 2002; Kudirkienė et al., 2010). However, in most studies one or two genotypes dominate in a flock while the remaining genotypes only are found sporadically. The presence of only one clone of C. jejuni in the present study may be explained by the ability of some clones to displace others in the chicken intestinal tract and colonize chickens consistently, as previously shown in the study of Calderon-Gomez et al. (2008). Besides, we only isolated Campylobacter from broiler cloacae by direct plating and additional genotypes may not be detected without an enrichment step if they were present in low numbers. The genotype detected during broiler rearing at the farm was also isolated in the slaughterhouse environment (surfaces of equipment, scalding water, and air), in agreement with other studies (Klein et al., 2007; Lienau et al., 2007; Newell et al., 2001) showing that Campylobacter is introduced into the slaughterhouse by Campylobacter positive broiler flocks. However, even though genotype VI dominated in the broiler cloacae and environmental samples at the slaughterhouse during processing, this particular genotype was only detected on one breast fillet sample at the end of the chain. It may be speculated that C. jejuni isolates belonging to this genotype could not survive the conditions during slaughtering such as low chilling temperature or high temperature of scalding water (Alter and Scherer, 2006; Humphrey et al., 2007; Murphy et al., 2006) and only a few cells survived until the end of processing. In the present study the dominant genotypes (I and IX) detected on broiler fillets were those also found in the slaughterhouse
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environment after disinfection before slaughter. Out of three different genotypes (I, VIII and IX) identified among isolates collected before slaughtering, genotype I together with genotype IX became dominant on broiler fillets. This is in sharp contrast to previous studies (Lienau et al., 2007; Takahashi et al. 2006), which showed that the final products were mainly contaminated with the genotypes carried by the broilers at the farm as well as genotypes isolated from slaughterhouse environment during processing and transport crates after cleaning and disinfection. It should however be noted that these studies did not include sampling of the environment of the slaughterhouse after disinfection and thus could not assess the importance of environmental contamination. It could be speculated that persistent isolates may have particular traits that allow survival in the slaughterhouse environment however; this needs to be confirmed by further characterization of the strains. To our knowledge only two additional studies have sampled and isolated C. jejuni in the environment of the slaughterhouse after disinfection (Johnsen et al. 2006; Peyrat et al. (2008a,b). Peyrat et al. (2008b) demonstrated that C. jejuni genotypes found after disinfection may contaminate broiler neck skin during slaughtering, however contamination with same genotypes from transport crates was not excluded in the study. Presently we cannot conclude whether the C. jejuni genotypes I and IX detected in our study had persisted in the slaughterhouse environment for a longer time period or if it just remained for a short while due to insufficient cleaning and disinfection procedures. Residues of organic matter, feathers on rubber fingers of the defeathering machine and scalding water were visually detected during sampling after disinfection, and these might increase the ability of C. jejuni to survive during disinfection procedure. In addition, failure of disinfection including too low concentration or too short application time of the disinfectants is possible. Interestingly, we were able to isolate C. jejuni from scalding water not only during processing, but also before the slaughtering, using an enrichment procedure for the isolation of Campylobacter spp. The presence of C. jejuni in scalding water has not been found in previous studies (Allen et al., 2007; Newell et al., 2001; Peyrat et al., 2008a), most likely because the isolation procedure used did not allow the detection of low numbers or sub-lethal damaged bacteria. Most strains of C. jejuni are susceptible to chlorine compounds (Blaser et al., 1986) and quaternary ammonium compounds (Peyrat et al., 2008a), the most commonly used disinfectant in broiler slaughterhouses. However, such strains may be protected from biocidal activities if they form a biofilm (Buswell et al., 1998) and the ability of other food borne pathogens like Listeria monocytogenes to survive in the slaughterhouse due to biofilm formation is well described (Gandhi and Chikindas, 2007). Recent studies carried out by Klein et al. (2007) and Takahashi et al. (2006) showed that even though C. jejuni isolates from the farm were highly diverse, only those genotypes that were most tolerant to environmental stresses were able to survive the slaughtering process and contaminate the final product (Klein et al., 2007). In agreement with this, the number of genotypes decreased notably from the beginning until the end of the slaughter line (Hiett et al., 2002; Klein et al., 2007). In contrast to this and other reports (Lindmark et al., 2006; Newell et al., 2001) we found several different C. jejuni genotypes on the final product and an increased diversity during the day. Only three different genotypes (I, II, and IX) were detected at the beginning of slaughter and after 4–6 h, while eight different genotypes (IX, III, II, IV, VII, V, VI, and X) could be found at the end of the day. Several factors may influence the results. First of all, the enrichment procedure was only used for environmental and not cloacae samples, which may bias the result as differences in genotypes detected using enrichment and direct plating were demonstrated by Newell et al. (2001). Secondly, the change of flaA genotype cannot be completely excluded as the reason of the rise of new genotypes on the breast fillets (de Boer et al., 2000). The
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intragenomic recombination between flaA and flaB genes between two strains resulting in appearance or disappearance of the new bands was demonstrated by Harrington et al. (1997). Against, the latter theory speaks that the conditions at the slaughterhouse are not favorable for growth of C. jejuni, therefore new genotypic variants do not occur frequently in the slaughterhouse. However, testing of genotypes found at the end of processing with another genotyping method(s) would be helpful to get optimal answer how the strains differ. 5. Conclusions The present study provides evidence that C. jejuni isolates present in the slaughterhouse environment after disinfection are an important source of broiler meat contamination. In contrast to previous studies, the genotypes of C. jejuni found on the surfaces of the equipment and in the scalding water of slaughterhouse after disinfection were also the dominating genotypes on broiler fillets, whereas contamination of broiler fillets with the C. jejuni genotype colonizing broilers during the rearing period was not significant in our study. Further characterization of the C. jejuni strains obtained along the entire meat production chain will be useful for our understanding of the survival mechanisms of C. jejuni in the adverse environment of the slaughterhouse, thus will allow us to improve the practices used to control C. jejuni in slaughterhouses. Acknowledgments This study was financially supported by the European Union funded Integrated Project BIOTRACER (contract FOOD-2006-CT036272) under the 6th RTD Framework. References Allen, V.M., Bull, S.A., Corry, J.E., Domingue, G., Jorgensen, F., Frost, J.A., Whyte, R., Gonzalez, A., Elviss, N., Humphrey, T.J., 2007. Campylobacter spp. contamination of chicken carcasses during processing in relation to flock colonisation. International Journal of Food Microbiology 113, 54–61. Alter, T., Scherer, K., 2006. Stress response of Campylobacter spp. and its role in food processing. Journal of Veterinary Medicine 53, 351–357. Alter, T., Gaull, F., Froeb, A., Fehlhaber, K., 2005. Distribution of Campylobacter jejuni strains at different stages of a turkey slaughter line. Food Microbiology 22, 345–351. Anonymous, 2010. The Community Summary Report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in the European Union in 2008. The EFSA Journal 1496, 124–127. Berndtson, E., Emanuelson, U., Engvall, A., Danielsson-Tham, M.L., 1996. A 1-year epidemiological study of campylobacters in 18 Swedish farms. Preventive Veterinary Medicine 26, 167–185. Blaser, M.J., Smith, P.F., Wang, W.L., Hoff, J.C., 1986. Inactivation of Campylobacter jejuni by chlorine and monochloramine. Applied and Environmental Microbiology 51, 307–311. Buswell, C.M., Herlihy, Y.M., Lawrence, L.M., McGuiggan, J.T., Marsh, P.D., Keevil, C.W., Leach, S.A., 1998. Extended survival and persistence of Campylobacter spp. in water and aquatic biofilms and their detection by immunofluorescent-antibody and rRNA staining. Applied and Environmental Microbiology 64, 733–741. Calderon-Gomez, L.I., Hartley, L.E., McCormack, A., Ringoir, D.D., Korolik, V., 2008. Potential use of characterised hyper-colonising strain(s) of Campylobacter jejuni to reduce circulation of environmental strains in commercial poultry. Veterinary Microbiology 134, 353–361. De Boer, P., Duim, B., Rigter, A., Van der Plas, J., Jacobs-Reitsma, W.F., Wagenaar, J.A., 2000. Computer-assisted analysis and epidemiological value of genotyping methods for Campylobacter jejuni and Campylobacter coli. Journal of Clinical Microbiology 38, 1940–1946. Gandhi, M., Chikindas, M.L., 2007. Listeria: a foodborne pathogen that knows how to survive. International Journal of Food Microbiology 113, 1–15.
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