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Discrepancy Between the Occurrence of Arcobacter in Chickens and Broiler Carcass Contamination

Department of Veterinary Public Health and Food Safety, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium

¹ Corresponding author: Kurt.Houf{at}UGent.be

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Both Campylobacter and Arcobacter are commonly present on broiler carcasses. For Campylobacter, the superficial contamination originates predominantly from fecal contamination during slaughter. In contrast with Campylobacter, the source of the Arcobacter contamination is not clear. In several studies, arcobacters have been isolated in poultry processing plants from the carcasses and slaughter equipment, but not from the intestinal content. In literature, contradictory reports about the Arcobacter colonization of the chicken gut have been published. In most of those studies, arcobacters were not isolated from cecal content nor from litter or the feathers, though some studies reported the isolation of arcobacters from cloacal swab samples. The present study assessed if arcobacters are part of the chicken intestine, skin, or feather flora. Because no isolation protocol has been validated for poultry intestinal content, a previously developed Arcobacter isolation procedure for feces from livestock animals was first validated. With this method, a good repeatability, in-lab reproducibility and sensitivity, and a good suppression of the chicken fecal accompanying flora were achieved when 125 mg/L of 5-fluorouracil, 10 mg/L of amphotericine B, 100 mg/L of cycloheximide, 16 mg/L of cefoperazone, 64 mg/L of novobiocine, and 64 mg/L of trimethoprim were applied. The validated method was used to examine the presence of arcobacters in and on living chickens of 4 flocks at slaughter age. Because arcobacters were not isolated from the intestinal tract nor from the skin or feathers of the birds, this study was not able to identify arcobacters as part of the intestinal or skin flora, nor could confirm the role of process water as reservoir. However, the results clearly demonstrated that the time period for processing the samples and the way of sample collection are crucial in the interpretation of epidemiological studies. As the reservoir of the carcass contamination remains unidentified, studies about the capacity of arcobacters to colonize the chicken intestinal tract may contribute in the assessment of the transmission routes of this emerging foodborn pathogen.

Key Words: poultry • Arcobacter • isolation method • host • natural chicken flora

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Bacteria belonging to the family Campylobacteraceae are important causes of human enteritis in most industrialized countries (Pearson et al., 2000). Two species, Campylobacter jejuni and Campylobacter coli, account for approximately 89% of human campylobacteriosis (Vandenberg et al., 2004). In 1992, some of the campylobacters, referred to as aerotolerant campylobacters, were transferred into the new genus, Arcobacter (Vandamme et al., 1991). Though arcobacters are closely related to campylobacters, they differ by their ability to grow in air and at lower temperature. At present, 4 species have been isolated from animal feces and food of animal origin: Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter skirrowii, and recently Arcobacter cibarius (Van Driessche et al., 2003; Rivas et al., 2004; Houf et al., 2005). The first 3 species have also been isolated from stool of humans with diarrheal illness, and in some cases septicemia (Wybo et al., 2003; Vandenberg et al., 2004). Similar clinical features as in a C. jejuni infection are displayed, but a higher association with persistent and watery diarrhea has been reported (Vandenberg et al., 2004). Recently, A. butzleri has been identified as the fourth most isolated Campylobacteraceae species from human diarrheal stool in Belgium and France (Vandenberg et al., 2004; Prouzet-Mauléon et al., 2006).

Consumption of food of animal origin and poultry products in particular has been identified as a major source of Campylobacter infection. Studies have shown that between 20 and 40% of human campylobacteriosis are due to the consumption of chicken meat (Nadeau et al., 2002; Vellinga and Van Loock, 2002). Also for Arcobacter, handling raw poultry, cross-contamination, and the consumption of undercooked poultry products have been pushed forward as potential infection sources (Corry and Atabay, 2001; Houf et al., 2002a). Biotyping and sero-typing studies have shown the distribution of the same types among human and poultry isolates (Lior and Woodward, 1993a,b; Marinescu et al., 1996).

Campylobacter and Arcobacter are commonly present on broiler carcasses (Harraß et al., 1998; Atabay et al., 1998; Corry and Atabay, 2001; Houf et al., 2001a, 2002a; Vytrasová et al., 2003; Houf et al., 2005). For Campylobacter, the superficial contamination originates predominantly from fecal contamination during the slaughter process, especially during plucking and evisceration (Rasschaert et al., 2006). The chickens in the broiler house become colonized in the cecum by horizontal transmission. Once introduced into the flock, Campylobacter spreads rapidly and the birds remain colonized until slaughter age (Newell and Fearnley, 2003). The Campylobacter carriage rates at flock level range from 18% to more than 90% depending on the region and isolation method applied (Kapperud et al., 1993; Evans and Sayers, 1997).

The prevalence of Arcobacter on Belgian poultry products was found higher than the prevalence of thermophilic Campylobacter species (Houf et al., 2001a). In contrast with Campylobacter, the source of the broiler carcass contamination is not clear. In several studies, arcobacters have been isolated in poultry processing plants from the carcasses and slaughter equipment, but not from the intestinal content (Houf et al., 2002b, Gude et al., 2005). Furthermore, enumeration and characterization of the isolates have shown that the slaughter equipment alone could not fully explain the high contamination levels on poultry products (Houf et al., 2002b; 2003). In literature, contradictory reports about the Arcobacter colonization of the chicken gut have been published. In most of those studies, arcobacters were not isolated from cecal content nor from litter or the feathers (Harraß et al., 1998; Houf et al., 2002b; Gude et al., 2005), though some studies reported the isolation of arcobacters from cloacal swab samples (Kabeya et al., 2003; Atabay et al., 2006). Infection experiments of chicks with A. butzleri were not successful (Wesley and Baetz, 1999; Eifert et al., 2003).

The aim of the present study was to assess if arcobacters are part of the chicken intestine, skin, or feather flora. Therefore, a previously developed Arcobacter selective isolation protocol was first validated for chicken feces. Then, the presence of arcobacters was determined in chickens from different flocks at slaughter age.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Validation of an Arcobacter Isolation Method
Because no method has been validated for the Arcobacter isolation from poultry intestinal content, Houf’s protocol was first validated in accordance with the validation procedure described by Van Driessche et al. (2003) for the isolation of arcobacters from feces of livestock animals. In brief, the same 6 Arcobacter reference strains were grown on blood agar plates (Mueller Hinton, CM 337, Oxoid, Basingstoke, UK, and 50 mL/L of lysed defibrinated horse blood [E&O Laboratories Ltd., Bonny-bridge, UK]) and incubated for 48 h at 28° under microaerobic conditions (6% CO₂, 6% H₂, 4% O₂, and 84% N₂). This incubation atmosphere was used for all further Arcobacter cultivations. From each strain, a bacterial suspension was prepared in 10 mL of sterile Arcobacter enrichment broth containing 24 g/L of Arcobacter broth (CM 965, Oxoid) and the previously adapted selective supplement (100 mg/L of 5-fluorouracil, 10 mg/L of amphotericine B, 16 mg/L of cefoperazone, 64 mg/L of novobiocine, and 64 mg/L of trimethoprim; Van Driessche et al., 2003) with a bacterial concentration of approximately 10⁹ cfu/mL. Serial 10-fold dilutions in sterile Arcobacter enrichment broth were prepared to obtain 10⁴ and 10³ cfu/mL for use in the validation of the direct isolation method and 10³ to 10⁰ cfu/mL for use in the validation of the enrichment method. Counts of the expected 10³ and 10⁴ cfu/mL bacterial suspensions were performed 10-fold by inoculating 100 µL by the spiral plating method onto Arcobacter selective agar plates (containing 24 g/L Arcobacter broth, 12 g/L Agar Technical No. 3 [L13-Oxoid] and the selective supplement described above). Following incubation at 28°C for 24 to 72 h, the average and the standard deviation of the logarithm of the colony counts were calculated.

For the direct isolation procedure, repeatability, in-lab reproducibility, sensitivity, and specificity were evaluated. For each of the 6 Arcobacter strains, 1 g of cecal content was taken in duplicate and transferred into sterile stomacher bags. Each sample was spiked respectively with 1 mL of the Arcobacter reference strain dilutions expected to be 10⁴ or 10³ cfu/mL. Then, 8 mL of Arcobacter enrichment broth was added, and the suspension was homogenized. Of each suspension, 20 samples of 100 µL were transferred onto Arcobacter selective agar plates by means of the spiral plater. The remaining homogenate was stored for 5 d at 4°C. After chill-storage, 100 µL was transferred 20 times onto Arcobacter selective agar plates. All agar plates were incubated for up to 5 d at 28°C and checked every 24 h for the presence of bacterial growth. The colonies were counted and identified by m-PCR (Houf et al., 2000). The average and standard deviation of the logarithm of the 20 colony counts were calculated.

For the enrichment method, the detection limit was determined. For each reference strain, 5 times 1 g of cecal content were taken and transferred into sterile stomacher bags. Eight milliliters of Arcobacter enrichment broth was added. Per strain, 4 samples were respectively spiked with 1 mL of the bacterial suspensions ranging from 10³ to 10⁰ cfu/mL. The fifth sample was spiked with 1 mL of sterile Arcobacter enrichment broth and acted as blank. After homogenization, all samples were incubated at 28°C. After 24, 48, and 72 h of incubation, 50 µL of the homogenates was streaked onto Arcobacter selective agar plates. The plates were incubated at 28°C and checked every 24 h for bacterial growth up to 3 d.

Results were analyzed using univariate ANOVA (P < 0.05).

Sample Collection at the Slaughterhouse
A Belgian poultry slaughterhouse that already participated in previous investigations (Houf et al., 2002b, 2003) was selected for this study. In this plant, the activities taking place in the living area (unloading, hanging, and killing of the birds) were separated from the rest of the processing activities to reduce the contamination of the evisceration room. The chickens slaughtered were between 35 and 42 d old. The slaughter capacity of the plant was approximately 8,000 chickens per hour, and 2 killing lines fed 1 evisceration line. The chickens were transported to the slaughterhouse in plastic crates with wire flooring that were slotted into pallets on large lorries. Upon their arrival at the processing plant, the coops with the birds were removed from the truck and placed on the floor pending further processing. The journey and holding time prior to slaughter were usually no more than a few hours. The birds were unloaded from the coops and directly hung on shackles. After electrical stunning and bleeding, the carcasses were scalded at 52°C for 180 s. The carcasses on each killing line passed through a single defeathering machine in which the feathers were removed by means of several rubber fingers on rotating disks coupled with copious water sprays. Evisceration was carried out along the processing line with a spoon-shaped scoop, and the internal organs were separated from the carcasses and presented for veterinary inspection. After the removal of the neck, crop, and lungs, the carcasses were washed inside and outside and air chilled before further distribution.

The slaughterhouse was visited on 4 d, and each time the first flock slaughtered on that day was sampled. Examined flocks of sampling d 1 to 4 are further referred to as flock 1 to 4. The statistical package Win Episcope 2.0 (Clive, Edinburgh, UK) was used to determine the maximum possible prevalence in percentage in function of the number of samples collected and the size of the flock. From flock 1 and flock 2 (both approximately 3,000 chickens), 10 living chickens per flock, waiting to be unloaded at the slaughter plant, were randomly collected. Euthanasia was carried out by cervical dislocation according to recommendations of the Center for Animal Welfare (undated), and the chickens were individually packed into sterile plastic bags. From flock 2, also 120 cloacal swab samples and 10 swabs from the transport crates were collected before onset of slaughter. During slaughter of the flocks, 10 neck skin samples were randomly collected immediately after defeathering. From flock 3 (approximately 6,000 birds), 15 neck skin samples and 300 intestinal tracts were randomly taken during slaughter and individually packed into sterile plastic bags. From flock 4 (approximately 5,000 birds), 10 neck skin samples and 250 intestinal tracts were collected during slaughter of the flock. Furthermore, water samples (500 mL) were collected from the scalding tank before and during the slaughter activities and from the water distribution pipe filling the scalding tank.

All samples were transported to the laboratory under cooled conditions (7°C) and processed within 4 h, except for 45 intestinal tracts of flock 4, which were processed the next day.

Sample Collection from Carcasses
From the 20 euthanized chickens collected from flock 1 and 2 before the onset of slaughter, samples of the feathers, neck, breast, and thigh skin, skin around cloaca, gall, spleen, liver, stomachs, crop, cecal, and cloacal content and from the small intestines were aseptically taken.

From the 300 intestinal tracts of flock 3, samples of 1 g were taken from the content of the small intestines and 1 cecum per tract, after cutting those parts of the intestines by means of sterile scissors.

To evaluate the external as well as the internal Arcobacter contamination, a swab sample from 205 intestinal tracts collected of flock 4 was taken from the ceca surfaces together with 1 g of content from 1 of the 2 ceca per tract. Then, the surface contamination of the other cecum was first eliminated as described by Rasschaert et al. (2006) by immersing the cecum in ethanol for 10 s. After drying by evaporation, a surface swab sample and 1 g of cecum content was taken.

To evaluate the effect of postponed sampling, samples from another 45 intestinal tracts of flock 4 were taken as described above after storage of the intestinal tracts for 24 h at 4°C.

Isolation of Arcobacter from Skin and Feather Samples
Arcobacters were isolated from the skin and feather samples using the isolation method by Houf et al. (2001a). In brief, for the enumeration of Arcobacters on 1 g of neck skin sample or feathers, 9 mL of Arcobacter enrichment broth was added to the sample homogenized in a stomacher blender. From each homogenate, 100 µL was brought onto an Arcobacter selective agar plate by the spiral plating method. To detect low contamination levels, the remaining homogenates were incubated microaerobically for 48 h at 28°C, followed by inoculation of 50 µL of the enrichment onto Arcobacter selective agar plates. All plates were incubated for 24 to 72 h at 28°C and checked every 24 h for bacterial growth. All colonies on the Arcobacter agar plates were counted, picked, and subcultured onto blood agar plates.

Isolation of Arcobacter from Swabs, Intestinal Content, and Internal Organs
The presence of arcobacters was investigated in 1 g of the internal organs of the dissected chickens, of all intestinal content samples, and in swabs of the transport crates and cloacal and intestinal surface swabs. Nine milliliters of Arcobacter enrichment broth (containing 24 g/L of Arcobacter broth, 50 mL/L of lysed defibrinated horse blood, and the adapted selective supplement) was added to the sample, and the mixture was gently shaken (swabs) or homogenized with a stomacher blender. The homogenates were incubated microaerobically for 48 h at 28°C, followed by inoculation of 50 µL of the enrichment onto Arcobacter selective agar plates (containing 24 g/L of Arcobacter broth, 12 g/L of Agar Technical No. 3, and the adapted selective supplement). All plates were incubated for 24 to 72 h at 28°C and checked every 24 h for bacterial growth.

Isolation of Arcobacter from Water Samples
The water samples (500 mL) were added to 500 mL double-strength Arcobacter enrichment broth as described by Houf et al. (2003). The homogenates were incubated microaerobically for 48 h at 28°C. After incubation, 50 µL of each homogenate was brought onto Arcobacter selective agar plates in quadruplicate. All plates were incubated for 24 to 72 h at 28°C and checked every 24 h for bacterial growth.

Identification and Characterization of Arcobacter Isolates
The DNA was prepared from the bacterial growth on the Arcobacter selective agar plates for the use in the identification and characterization assays. Therefore, 1 colony of each plate was suspended in 500 µL of sterile distilled water and boiled for 10 min. After a quick spin, 2 µL of the supernatant was used as DNA template.

For identification of the isolates, an Arcobacter species-specific multiplex PCR assay (Houf et al., 2000) was performed in a reaction mixture of 50 µL final volume, using the primers SKIR, ARCO, BUTZ, CRY1, and CRY2 (In-vitrogen, Paisley, Scotland).

A selection of the obtained isolates was characterized below species level by ERIC-PCR (Houf et al., 2002a). In brief, 1 µL of DNA was added to the 49-µL PCR volume. The ERIC motifs 1R 5'-ATGTAAGCTCCTGGGGATT-CAC-3' and 2 5'AAGTAAGTGACTGGGGTGAGCG-3' were used at concentrations of 25 pmol each. The PCR products were size-separated by electrophoresis in 2% agarose gels. The banding pattern used to determine the ERIC-PCR type comprised DNA fragments between 100 and 2,072 bp. Computer-based normalization and interpolation of the DNA profiles, and numerical analysis using the Pearson product moment correlation coefficient, with 1% position tolerance, were performed using the GelCompar 4.2 software package (Applied Maths, Kortrÿk, Belgium). Dendrograms were constructed using the unweighted pair group linkage analysis method. For convenience, the correlation level was expressed as a percentage similarity. As shown in previous studies, DNA patterns that differed in 1 or more DNA fragments represented different types (Houf et al., 2002b, 2003).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Validation of the Isolation Medium
Arcobacter was not isolated from the samples used in the validation tests as blanks. With the direct isolation method, the recovery of the different Arcobacter species at both spiking levels was concordant with the initial number added (P < 0.05). The standard deviation on the colony counts ranged from 0.02 to a maximum of 0.11. There was no interspecies nor intraspecies difference in recovery, only a difference in the time required for outgrowth. For A. butzleri, 24 h incubation was already sufficient to obtain visible colonies, whereas 48 and 72 h were necessary for A. cryaerophilus and A. skirrowii, respectively. To determine the in-lab reproducibility, the spiked homogenates were stored for 5 d at 4°C. Colony counts were still between 10² to 10³ and 10³ to 10⁴ cfu/mL for the homogenates spiked with 10³ and 10⁴ cfu/mL, respectively, but had significantly decreased (P < 0.05) in comparison with the colony counts for the direct isolation. For the enrichment method, the detection limit was set on 10⁰ cfu/g. At this concentration, A. butzleri could be isolated after 24 h of enrichment, followed by 24 h of plate incubation. Arcobacter cryaerophilus and A. skirrowii required an enrichment of 48 h followed by 48 h of plate incubation. An enrichment of 72 h was not required for any of the Arcobacter species tested. Growth of accompanying flora was observed on some plates. Therefore, based on the minimal inhibitory concentration study of Houf et al. (2001b), the concentration of 5-fluorouracil was increased to 125 mg/L, which improved the specificity of the isolation medium. The adapted selective supplement for the Arcobacter isolation from chicken intestinal content contains 125 mg/L of 5-fluorouracil, 10 mg/L of amphotericine B, 100 mg/L of cycloheximide, 16 mg/L of cefoperazone, 64 mg/L of novobiocine, and 64 mg/L of tri-methoprim.

Presence of Arcobacter in the Samples
For all 4 flocks, all neck skin samples taken along the slaughterline prior to evisceration, were contaminated with arcobacters at numbers of >10³ cfu/g of skin. Species distribution among the neck skin samples positive for Arcobacters were given in Table 1. The ERIC-PCR distinguished 15 different genotypes among the 18 A. butzleri neck skin isolates of flock 4.

From flocks 1 and 2, 10 birds were euthanized and dissected. Results of the Arcobacter isolations after enrichment of internal organs, skin and feather samples of these birds are given in Table 2. No arcobacters were detected in the 120 cloacal swabs taken from flock 2. Nevertheless, arcobacters were isolated from 7 of the 10 swab samples taken from the transport crates of flock 2, and 20 isolates were identified as A. butzleri (n = 2) and A. cryaerophilus (n = 18).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The origin of the almost ubiquitous presence of arcobacters on the carcasses of broilers, but also on laying hens, is still under discussion because the transmission routes of these bacteria are still not established. In contrast to the closely related Campylobacter, for which the contamination at broiler house level is well documented and easily detected by conventional microbiological methods, arcobacters seem to display a different behavior.

One of the aspects that actually hamper the risk assessment for these bacteria is that most isolations are performed by methods designed for thermophilic Campylobacter species or Yersinia or even Leptospira (Houf et al., 2001a). The selective isolation in those media is achieved by the incorporation of antimicrobial agents in the plating media. However, a study about the Arcobacter susceptibility to antimicrobials included in those media revealed that none of those selective supplements allowed the growth of all Arcobacter strains and at the same time sufficiently suppressed the accompanying flora in biological samples (Houf et al., 2001b). A second isolation strategy includes the filtration of a feces homogenate onto nonselective blood agar plates, as used by Lastovica and Le Roux (2000). Though theoretically this method allows isolation of more sensitive species, it has not been validated for Arcobacter isolation. Furthermore, selective isolation based on filtration applied in food microbiology has already shown to be less sensitive compared with selective isolation based on incorporation of antimicrobial agents (Houf et al., 2001a; Gude et al., 2005). In the present study, a direct isolation method and enrichment protocol for the Arcobacter isolation from feces of livestock was validated for the isolation of Arcobacter species from poultry intestinal content. Using the validated method, poultry intestinal samples were examined. Although arcobacters were isolated from all neck skin samples taken along the processing line, arcobacters have only been isolated from 1 crop, 1 cloacal swab, and 1 skin sample from the dissected chickens. These results confirm the findings of Harraß et al. (1998) and Houf et al. (2002b), in which no arcobacters were detected in 170 cecal samples and 30 intestinal tracts, respectively, although skin samples of the same flock at slaughter were contaminated. Also in the study of Atabay and Corry (1997), which screened 15 abattoir carcasses (skin, gizzard, small intestine, cecum, and colon), arcobacters were isolated from the skin of every carcass, but Arcobacter was only detected in the colon of 1 birth. These authors suggested also that arcobacters were probably not normal inhabitants of the poultry intestine and that, as previously formulated by Eifert et al. (2003), Houf et al. (2003), and Gude et al. (2005), process water may be a potential source of the carcass contamination. In the study by Wesley and Baetz (1999), 407 cloacal swabs were examined by arcobacters and were isolated from 15% of samples. Nevertheless, when the age of the chickens was taken into account, the highest prevalence was detected in 56-wk-old chickens (57%), whereas only 1 from the 105 samples of chickens at slaughter age (5 to 8 wk) was positive for the presence of Arcobacter.

Considering that in the study of Houf et al. (2003) and in others almost all broiler carcasses, even the very first carcass slaughtered after some days of nonactivity in the slaughterhouse, were contaminated with arcobacters at levels of 100 and more bacteria per gram of skin, a continuous source of arcobacters at a high level is needed. In the present study, the size of samples from flocks 2 to 4 allowed the detection of an Arcobacter prevalence in the intestines of approximately 1%. Assuming that the carcass contamination originates from fecal contamination, the prevalence of 1% in the intestines of chickens at slaughter age can hardly be the source of carcass contamination of 10¹ to 10⁴ cfu/g of skin of the whole flock. Though statistically possible, it should mean that only a minority of birds carry an enormous level of arcobacters, which would not only differ from the closely related Campylobacter behavior but furthermore cannot explain why the first carcasses slaughtered were already contaminated.

Besides the isolation method, another explanation for the contradictory reports in literature can be caused by the way of sampling. In the present study, arcobacters were isolated from the intestinal samples of 550 intestinal tracts of flock 3 and 4, but when surface disinfection was applied in flock 4, no arcobacters were isolated. The ethanol treatment has previously been applied in Campylobacter research, and also in this study, it had no impact on the possible arcobacters present in the intestine lumen because arcobacters were detected in the ethanol-treated cecum from the intestinal tracts sampled after 24 h. That all the intestinal tracts sampled 24 h postslaughter were positive for arcobacters makes it likely that tissue texture was lost and that bacteria could migrate from the surface to the intestine content. Because in the present and in previous studies arcobacters were isolated from the crates to transport the chickens to the slaughterhouse, one should take into account that those arcobacters may contaminate the cloacal region and may explain the isolations reported by Kabeya et al. (2003) and Atabay et al. (2006). Both observations in the present study clearly demonstrated that the time and the procedure of sampling is crucial and can affect the outcome of the study and the conclusions.

Because arcobacters were not isolated from the intestinal tract or from the skin or feathers of the birds, the hypothesis of process water as the source of arcobacter contamination is interesting to investigate. Also in the present study, arcobacters were isolated from different water samples taken in the slaughterhouse and even the fresh water used in the scalding tanks. Nevertheless, characterization of the isolates could not confirm this source due to the large heterogeneity among the isolates.

In conclusion, the results of the present study have clearly demonstrated that the time period for processing the samples and the way of sample collection are crucial in the study about the contamination source of arcobacters. The results could not identify arcobacters as part of the intestinal or skin flora of chickens or confirm the role of process water as reservoir. This is in contrast with the Campylobacter epidemiology in chickens and also with the contamination routes of arcobacters in pigs and cattle (Van Driessche, 2004, 2005; Ho et al., 2006). As demonstrated in the studies by Houf et al. (2002a, 2003), characterization of a large amount of isolates does not contribute to a better understanding of Arcobacter epidemiology due to the large heterogeneity among those bacteria. Therefore, studies about the capacity of arcobacters to colonize the chicken intestinal tract may contribute in the assessment of the epidemiology of this emerging foodborne pathogen.

	ACKNOWLEDGMENTS

 
This work was supported by the Research Fund of the Ghent University, Belgium, codenr. BOF 2002/DRMAN/ 063. We thank the BCCM/LMG Bacteria Collection, Ghent University (Ghent, Belgium) for providing the Arcobacter collection strains used in this study.

Received for publication September 1, 2006. Accepted for publication December 4, 2006.

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