Quantitative estimation of Campylobacter cross-contamination in carcasses and chicken products at an abattoir

Food Control 43 (2014) 10e17

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Quantitative estimation of Campylobacter cross-contamination in carcasses and chicken products at an abattoir Yoshimasa Sasaki a, *, Mika Haruna a, Tetsuya Mori b, Masashi Kusukawa a, Mariko Murakami a, Yayoi Tsujiyama a, Kazuo Ito a, Hajime Toyofuku c, Yukiko Yamada a a b c

Food Safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry and Fisheries, 1-2-1 Kasumigaseki, Chiyoda-ku, Tokyo 100-8950, Japan Institute for Food and Environment Sciences in Tokyo Kenbikyo-in Foundation, 5-1 Toyomi-cho, Chuo-ku, Tokyo 104-0055, Japan Department of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida Yamaguchi-shi, Yamaguchi 753-8515, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 May 2013 Received in revised form 5 February 2014 Accepted 11 February 2014

This study aimed to investigate Campylobacter contamination in carcasses and chicken products derived from a Campylobacter-negative flock when the flock is slaughtered immediately after a Campylobacterpositive flock. The first 2 flocks slaughtered on 10 different dates were investigated at an abattoir. Eighteen of the 20 flocks tested were positive for Campylobacter. A Campylobacter-negative flock was slaughtered immediately after a Campylobacter-positive flock on only 1 of the 10 slaughter dates. In this case, Campylobacter was detected in the carcasses and chicken products originating from the Campylobacternegative flock, and all the flaA genotypes of these isolates were identical to those present in the caecal contents, carcasses, and chicken products from the Campylobacter-positive flock. The Campylobacter concentrations in the products originating from the Campylobacter-negative flock were: close to the enumeration limit (1.7 log10 cfu/carcass) in the carcass samples; and below the enumeration limit (2.0 log10 cfu/g) in the liver samples. The mean Campylobacter concentrations in the carcasses and liver products originating from the 18 Campylobacter-positive flocks were 3.8 log10 cfu/carcass and 2.6 log10 cfu/ g, respectively. While 91% (246/270) of chicken products originating from Campylobacter-positive flocks were positive for Campylobacter, chicken products originating from the remaining Campylobacter-negative flock were free from Campylobacter cross-contamination by slaughter prior to a Campylobacter-positive flock. These results prove that slaughtering Campylobacter-negative flocks does not introduce Campylobacter into the abattoirs and indicate that although carcasses and chicken products originating from the Campylobacter-negative flock were cross-contaminated with Campylobacter from the Campylobacterpositive flock slaughtered immediately before, the Campylobacter contamination levels were lower than those in carcasses and chicken products from Campylobacter-positive flocks. Based on these findings, the reduction of Campylobacter prevalence in broiler flocks should be taken as an effective control measure for preventing introduction of Campylobacter into abattoirs and consequently for reducing Campylobacter prevalence in chicken products in addition to the good hygienic practice at abattoirs and logistic slaughter. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Campylobacter cross-contamination Carcass Chicken product Abattoir

1. Introduction Human campylobacteriosis is one of the most important foodborne diseases worldwide, and consumption of raw or undercooked poultry meat contaminated with Campylobacter has been identified as a risk factor for such an illness (EFSA Panel on Biological Hazards, 2011; Kapperud, Skjerve, Bean, Ostroff, & Lassen, 1992; Pearson et al., 2000). Kubota et al. (2011) estimated that 1210 cases of foodborne

* Corresponding author. Tel.: þ81 3 3502 5722; fax: þ81 3 3597 0329. E-mail address: [email protected] (Y. Sasaki). http://dx.doi.org/10.1016/j.foodcont.2014.02.015 0956-7135/Ó 2014 Elsevier Ltd. All rights reserved.

campylobacteriosis per 100,000 population occurred each year in Miyagi Prefecture in Japan. In 2009, risk assessment conducted by the Food Safety Commission (FSC) in Japan for Campylobacter in poultry indicated the following most effective risk management options: (i) slaughter of Campylobacter-negative flocks before Campylobacter-positive flocks (logistic slaughter) and (ii) maintenance of an adequate concentration range (1e5 mg/L) of free available chlorine (FAC) in chilled water tanks (FSC, 2009). These 2 approaches can reduce the risk of human Campylobacter infection by 44% and 21%, respectively. The risk assessment used the study of a probabilistic risk assessment model for Campylobacter infection from poultry meat and it estimated that approximately 100 million people

Y. Sasaki et al. / Food Control 43 (2014) 10e17

were infected with Campylobacter per year in Japan (Kasuga, 2011). In contrast, Nauta et al. (2009) reviewed several quantitative risk assessments of Campylobacter in broiler meat conducted in countries other than Japan and concluded that logistic slaughter has a negligible effect on the reduction of campylobacteriosis risk in all risk assessments. The different conclusions regarding the effect of logistic slaughter may be attributed to several data gaps in the risk assessment carried out in Japan. For example, neither Campylobacter prevalence nor concentration in chicken products originating from Campylobacter-negative flocks cross-contaminated by Campylobacter-positive flocks during the slaughtering process were used for the risk assessment conducted by FSC. A more refined assessment will be possible when these data gaps are filled. Therefore, we had previously investigated whether chicken products originating from a Campylobacter-negative flock become cross-contaminated by a Campylobacter-positive flock that was slaughtered immediately before, at an abattoir in Japan. Our results showed that 198 out of 600 (33.0%) bulk packed chicken products originating from 24 broiler flocks were contaminated with Campylobacter and that 14 chicken

11

products originating from a Campylobacter-negative flock were cross-contaminated by a Campylobacter-positive flock during the slaughtering process (Sasaki et al., 2012). However, the Campylobacter concentration in chicken products cross-contaminated by the Campylobacter-positive flock was not investigated in that study. Since quantitative data will be necessary and useful for conducting a quantitative risk assessment, the present study aimed to obtain the concentration of Campylobacter in carcasses and chicken products cross-contaminated by the Campylobacter-positive flock that was slaughtered immediately before the flock was tested. 2. Materials and methods 2.1. Slaughtering and processing of broilers A broiler production company voluntarily participated in this study. The abattoir was located in the Kanto region of the eastern part of Japan. Fig. 1 summarizes the flow of broiler processing in the abattoir. After processing of broilers, 2 kg of each chicken breast

Fig. 1. Flow diagram of broiler processing in the abattoir investigated. TC, the total chlorine concentration.

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Y. Sasaki et al. / Food Control 43 (2014) 10e17

with skin, tender, or liver were packed in plastic bags, stored in a cold room at 4  C  1  C and shipped to the market within a few days of processing. 2.2. Sampling Sampling was performed at the abattoir on 10 different slaughter dates between September 2010 and February 2011 (Table 1). On each of these dates, the first 2 flocks slaughtered on that day were selected for sampling. A flock was defined as a group of birds raised in the same broiler house during the same period of time. In total, 20 flocks, designated as A to T, from 11 broiler farms, designated as FA to FK, were tested. Eight flocks (A, D, E, G, I, M, O and S), 2 flocks (F and P), and 2 flocks (K and Q) were obtained from FA, FD and FG, respectively. The mean flock age at slaughter was 51 days (minimum and maximum, 48 and 54 days, respectively). After evisceration, 10 caecal content samples (approximately 5 g for each bird) were collected from 10 individual broilers of each selected flock and placed in an anaerobic Kenki-porter vial (Terumo Clinical Supply, Gifu, Japan). Five carcasses were collected from each selected flock after the second immersion in chilled chlorinated water. Each carcass was kept in a plastic bag. At the beginning, the middle, and the end of the second immersion of the carcasses, chilled water samples were collected in sterilized 1.0 L bottles, and the concentration of FAC was measured using the Pocket Colorimeter II Kit (Hach Co., Loveland, Colorado, USA). To neutralize residual chlorine, several sodium thiosulphate crystals were then added to the bottles. Subsequently, each 5 packs (2 kg/pack) of breasts with retained skin, tenders (supracoracoideus), and livers were collected. Samples were transported to the Institute for Food and Environment Sciences, Tokyo Kenbikyo-in Foundation, via express delivery under refrigeration. At the laboratory, samples were refrigerated at 4  C  2  C and tested within 24 h after each arrival.

2.3. Isolation and enumeration of Campylobacter and total aerobic bacteria 2.3.1. Caecal content samples For detecting Campylobacter, approximately 0.1 g of each sample was plated directly onto modified charcoal cefoperazone deoxycholate agar (mCCDA) (Oxoid, Basingstoke, Hampshire, England) and incubated at 42  C for 48 h under a microaerobic atmosphere generated using an AnaeroPack-MicroAero (Mitsubishi Gas Chemical Co., Ltd., Tokyo, Japan). Meanwhile, 1 g of the remaining sample was mixed with 9 mL of Preston broth (Oxoid) and incubated at 42  C for 24 h to produce enrichment cultures. A loopful (ca. 0.1 mL) of Preston broth was then plated onto mCCDA and incubated under a microaerobic atmosphere at 42  C for 48 h. Suspect colonies (3 per plate) were picked and cultured by plating onto mCCDA and nutrient agar (NA) (Nissui Pharmaceutical, Co., Ltd., Tokyo, Japan). Enumeration of Campylobacter in caecal samples was performed by serial dilutions (1:10) in Preston broth, and each diluted suspension was plated in duplicate on the surface of mCCDA plates. After incubation for 48 h at 42  C, colony-forming units (cfu) of Campylobacter were counted. Finally, 2 suspect colonies were cultured by plating onto mCCDA and NA for further identification of Campylobacter. The mean of duplicate counts was calculated and converted to log10 cfu/g of caecal content samples. The enumeration limit of the method was 2.0 log10 cfu/g. When a sample was negative by the enumeration method but positive by direct plating and/or enrichment, the sample was given a value of 1.0 log10 cfu/g, one-half of the enumeration limit. 2.3.2. Carcasses Each Carcass placed in a sterile bag was rinsed manually for 2 min with 550 mL of buffered peptone water, 500 mL of which was then centrifuged at 8000  g for 30 min at 4  C. After the supernatants were carefully removed, 5 mL of distilled water (DW) was added to the precipitates. DW suspensions (1 mL) were

Table 1 Concentration of Campylobacter in caecal contents, carcasses and chicken products. Day of slaughter

Flocka

Caecal content b

7 September 2010 28 September 2010 19 October 2010 26 October 2010 16 November 2010 30 November 2010 7 December 2010 18 January 2011 15 February 2011 22 February 2011 Total a b c d e

A B C D E F G H I J K L M N O P Q R S T

Carcass

Breast

N

Mean log10 cfu/gc  SDd

N

Mean log10 cfu/carcass  SD

10 10 10 10 10 10 10 10 10 0 10 10 10 10 6 10 0 10 10 10 176/200

5.1  6.4  5.6  6.3  6.5  7.2  6.9  6.2  5.4  NDe 5.0  5.8  4.9  4.3  4.3  5.0  ND 5.8  5.8  5.9 

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 100/100

3.0 5.9 2.4 4.0 4.7 4.4 4.2 3.5 4.2 1.8 4.2 4.1 4.9 3.7 2.2 3.6 0.9 3.3 3.4 3.4

0.8 0.7 1.0 0.6 0.8 0.4 0.6 1.0 1.3 1.2 1.3 0.8 1.6 0.2 0.6 1.0 0.6 0.9

               

0.3 0.6 0.4 0.7 0.9 1.0 0.8 0.2 0.5 0.5 0.7 0.3 1.0 0.5 0.5 0.8

 0.8  0.5  0.7

Tender

Liver

N

Mean log10 cfu/g  SD

N

Mean log10 cfu/g  SD

N

Mean log10 cfu/g  SD

5 5 5 5 5 5 5 5 5 1 5 5 5 5 5 4 0 5 5 5 90/100

1.0 1.7  1.0 1.6  1.9  2.2  1.8  2.2  1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 ND 2.0  1.2  1.6 

4 5 1 5 4 5 3 5 1 2 4 4 5 2 0 4 0 5 5 5 69/100

1.0 1.4  2.0 1.0 1.3  1.5  1.3  1.4  1.0 1.0 1.0 1.0 1.0 1.0 ND 1.3  ND 1.2  1.0 1.4 

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 0 5 5 5 95/100

2.2  3.3  3.3  2.4  2.7  2.8  2.5  2.8  2.2  1.0 2.2  2.7  3.5  2.1  2.4  2.5  ND 2.5  2.8  2.6 

0.6 0.5 0.5 0.2 0.4 0.1

0.6 0.5 0.5

On one day a flock in the top row was slaughtered before the other flock in the bottom row. The number of positive samples among 10 samples (caecal content) or 5 samples (carcass, breast, tender and liver) taken. Mean of positive samples. Standard deviation. Not detected.

0.5

0.5 0.6 0.6 0.5

0.5 0.5 0.5

0.2 0.6 1.2 0.1 0.5 0.3 0.3 0.2 0.9 0.2 0.3 1.1 0.2 0.5 0.4 0.2 0.6 0.4

Y. Sasaki et al. / Food Control 43 (2014) 10e17

subsequently mixed with 9 mL of Preston broth and incubated at 42  C for 24 h to produce enrichment cultures. After incubation, a loopful (ca. 0.1 mL) of the resulting Preston broth culture was plated onto mCCDA and incubated under a microaerobic atmosphere at 42  C for 48 h. Suspect colonies (3 per plate) were picked and cultured by plating onto mCCDA and NA. Enumeration of Campylobacter in DW suspensions was performed as described above and the counts were converted to log10 cfu/carcass. The enumeration limit of the method was 1.7 log10 cfu/carcass. When a sample was negative by the enumeration method but positive by enrichment, the sample was given a value of 0.9 log10 cfu/carcass, one-half of the enumeration limit. 2.3.3. Chicken products Each sample (25 g) consisted of 5 pieces (5 g each) of meat in a pack. After each sample was stomached in 225 mL of Preston broth, 1 mL was used for the enumeration of Campylobacter, and the remaining was incubated at 42  C for 24 h for enrichment. After incubation, a loopful (ca. 0.1 mL) of the resulting Preston broth culture was then plated onto mCCDA and incubated under a microaerobic atmosphere at 42  C for 48 h. Suspect colonies (3 per plate) were picked and cultured individually by plating onto mCCDA and NA. Enumeration of Campylobacter was performed as described above. The enumeration limit of the method was 2.0 log10 cfu/g of chicken products. When a sample was negative by the enumeration method but positive by enrichment, the sample was given a value of 1.0 log10 cfu/g, one-half of the enumeration limit. 2.3.4. Chilled water samples For detecting Campylobacter, 1 L chilled water was centrifuged at 8000  g for 30 min at 4  C. After the supernatants were carefully removed, 5 mL of DW was added to the precipitates. DW suspensions (1 mL) were then mixed with 9 mL of Preston broth and incubated at 42  C for 24 h for enrichment. Next, a loopful of the resulting Preston broth culture was plated onto mCCDA and incubated under a microaerobic atmosphere at 42  C for 48 h. Suspect colonies (3 per plate) were picked and cultured by plating onto mCCDA and NA. Enumeration of Campylobacter was performed as described above. When Campylobacter was only detectable after enrichment, its concentration was considered to be unquantifiable positive (þ). Enumeration of total aerobic bacteria (TAB) was performed by serial dilution (1:10) in DW. Samples (0.1 mL) from serial dilutions were plated in duplicate on the surface of standard methods agar medium (Nissui Pharmaceutical Co., Japan). After incubation at 36  C for 24 h, colony-forming units of TAB were counted. The mean of duplicate counts was calculated and converted to log10 cfu/mL. 2.4. Identification of Campylobacter Suspect colonies were subjected to Gram staining, as well as tests for catalase and oxidase production, motility, and indoxyl acetate hydrolysis (On, 1996). Hippurate hydrolysis and the Campylobacter (cdt gene) PCR Detection and Typing Kit (Takara Bio Inc., Tokyo, Japan) were used to identify Campylobacter jejuni and Campylobacter coli. 2.5. flaA typing of Campylobacter isolates A maximum of 2 C. jejuni and 2 C. coli isolates obtained from each caecal content, carcass, chicken product, and chilled tank water sample were delivered to a laboratory of the Japan Food Research Laboratories (JFRL) and characterized by flaA typing (Nachamkin, Bohachick, & Patton, 1993).

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3. Results 3.1. Prevalence and concentrations of Campylobacter in caecal contents, carcasses, chicken products, and chilled water As shown in Table 1, Campylobacter was detected in caecal content samples collected from 18 of 20 (90%) broiler flocks, and therefore, these 18 flocks were considered to be Campylobacterpositive. In 17 of the 18 positive flocks, Campylobacter was detected in all of the 10 caecal samples tested for each flock. The mean log10 value of Campylobacter concentrations of 176 caecal content samples from Campylobacter-positive flocks was 5.7 log10 cfu/g, and 95.5% (168/176) of these caecal contents had more than 4.0 log10 cfu/g Campylobacter. Two flocks, J and Q, were considered to be Campylobacter-negative since no Campylobacter was isolated from any caecal contents of these flocks. Campylobacter was isolated from all the carcasses, including those originating from the 2 Campylobacter-negative flocks. Campylobacter was isolated from all the carcasses from the 18 Campylobacter-positive flocks without enrichment and the mean concentration of Campylobacter was 3.8 log10 cfu/carcass. As for the carcasses from flock J, without enrichment, Campylobacter was detected only close to the enumeration limit in 4 of 5 carcasses tested and not detected in the remaining one carcass. The mean Campylobacter concentration was estimated to be close to the enumeration limit. Campylobacter was also present in carcasses from flock Q, but the level was only detectable after enrichment (0.9 log10 cfu/carcass). With regard to chicken products, Campylobacter was isolated from 254 (85%) of 300 chicken products. The prevalence rates of Campylobacter in chicken products originating from Campylobacterpositive and -negative flocks were 91% (246/270) and 27% (8/30), respectively. The prevalence rate of Campylobacter in tender products (69%, 69/100) was significantly lower than that in either breast (90%, 90/100) or liver (95%, 95/100) products (P < 0.001, Fisher’s exact test). In addition, only after enrichment was Campylobacter detectable in 63% (57/90) and 81% (56/69) of the breast and tender products, respectively. Of 90 Campylobacter-positive breast products, 89 were derived from Campylobacter-positive flocks, with the remaining one from flock J. Based on these data, the prevalence of Campylobacter in breast products from the Campylobacter-negative flock (flock J) (20%, 1/5) was significantly lower than that from the 18 Campylobacter-positive flocks (99%, 89/90) (P < 0.001, Fisher’s exact test). Out of the 69 Campylobacter-positive tender products, 67 (97%) were derived from the 17 Campylobacter-positive flocks, and the remaining 2 were from flock J. No Campylobacter was detected in the tender products from 2 flocks, O and Q. Among these 2 flocks, flock Q was Campylobacter-negative and flock O was one of 2 Campylobacter-positive flocks with the lowest mean Campylobacter concentration in caecal content (4.3 log10 cfu/g). The prevalence of Campylobacter in the tender products from all of the second flocks slaughtered (84%, 42/50) was significantly higher than that in all of the first flocks slaughtered (54%, 27/50) (P < 0.001, Fisher’s exact test) in the abattoir. In the case of liver products, all products originating from the 18 Campylobacter-positive flocks were positive for Campylobacter, and the mean Campylobacter concentration in these products was 2.6 log10 cfu/g. In addition, Campylobacter was also detected in all of the liver products from flock J, but only after enrichment (considered as 1.0 log10 cfu/g). No Campylobacter was present in any liver products obtained from flock Q. All the 60 chilled water samples taken from the chill tank contained FAC, and the concentrations ranged from 0.2 to 24.0 mg/L (Table 2). Among these samples, 25 (42%) were found to be

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Y. Sasaki et al. / Food Control 43 (2014) 10e17

Table 2 Concentration of Campylobacter, total aerobic bacteria (TAB) and free available chlorine (FAC) in chill tank water. Day of slaughter

Flock

Timing of water sampling

Campylobacter (log10 cfu/200 mL)

TAB (log10 cfu/mL)

FAC (mg/L)

7 September 2010

A

Beginning Middle End Beginning Middle End Beginning Middle End Beginning Middle End Beginning Middle End Beginning Middle End Beginning Middle End Beginning Middle End Beginning Middle End Beginning Middle End Beginning Middle End Beginning Middle End Beginning Middle End Beginning Middle End Beginning Middle End Beginning Middle End Beginning Middle End Beginning Middle End Beginning Middle End Beginning Middle End

e þ 1.5 þ 2.3 2.5 e e þ 1.0 1.6 1.0 e e e þ 1.3 1.5 e 1.0 e e 1.0 1.0 e e e e e e e þ e e e e e þ e e e e e e e e e e e e e þ 2.7 2.5 e 1.0 1.0 þ 1.8 1.0

e 1.9 1.6 2.2 2.1 2.5 e e 1.2 2.7 2.8 2.7 0.5 e 1.5 1.5 1.9 2.5 e e 1.2 2.7 2.8 2.7 e e e 0.5 2.3 e e e e e e e e e e e e 0.3 e e e e 0.8 1.3 e e 2.3 2.4 2.1 2.4 e e 1.6 1.6 2.3 2.3

9.0 1.0 11.0 3.0 3.0 4.0 0.4 2.2 0.2 0.5 0.3 0.2 1.3 1.0 2.0 7.0 2.2 1.0 2.0 4.0 2.1 0.5 2.6 2.1 2.0 7.0 4.0 3.0 3.0 5.0 12.0 16.0 13.0 11.0 15.0 18.0 18.0 24.0 23.0 21.0 15.0 17.0 15.0 11.0 3.0 1.0 1.0 4.0 8.0 1.0 1.6 1.0 2.0 1.0 4.0 1.0 0.8 1.0 1.0 1.0

B

28 September 2010

C

D

19 October 2010

E

F

26 October 2010

G

H

16 November 2010

I

J

30 November 2010

K

L

7 December 2010

M

N

18 January 2011

O

P

15 February 2011

Q

R

22 February 2011

S

T

e: Not detected, þ: colonies were obtained only after enrichment culture.

Campylobacter-positive, of which 17 (68%) were from the chill tank where the second flocks slaughtered were soaked. The maximum concentration of Campylobacter was 2.7 log10 cfu/200 mL chilled water. Moreover, TAB were detected in 31 (52%) samples, of which 23 (74%) were from the chill tank where the second flocks slaughtered was soaked. The TAB concentration was over 2.0 log10 cfu/mL in 17 samples, and 16 (94%) of which were from the chill tank where the second flocks slaughtered was soaked. For all the 12 samples obtained on 30 November and 7 December 2010, as

a result of the FAC concentrations being higher than 10 mg/L, Campylobacter and TAB were only detected in 2 (17%) and 1 (8%) samples, respectively. 3.2. flaA genotyping of Campylobacter isolates To characterize the Campylobacter isolates obtained in the present study, flaA genotyping was conducted based on the fact that there are 12 and 4 restriction fragment length polymorphism

Y. Sasaki et al. / Food Control 43 (2014) 10e17

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Table 3 flaA genotypes of Campylobacter isolates. Day of slaughter

7 September 2010 28 September 2010 19 October 2010 26 October 2010 16 November 2010 30 November 2010 7 December 2010 18 January 2011 15 February 2011 22 February 2011

Flock

A B C D E F G H I J K L M N O P Q R S T

Caecal content

Chill tank water

Carcass

Breast

Tender

Liver

Isolate

Isolate

Isolate

Isolate

Isolate

Isolate

J2, C1, C2 J10, C1 J8 J8, C1 J1 J10, C1 J1 J8 J5, J8, C1 e J6, J11 J1 J8 J1 J8, J9 J8 e J4 J8 J4, J8

J2 J10 J5 J1, J12 e J10 J1 J8 e e J6 e J8 e e e e J4 J8 J8

J2 J10 J8, J12 J8 J1 J5, J10 J1 J1, J8, C1 J2, C1 J2, J8 J6, J11 J1 J5 J1 J8 J8 J4, J12 J4 J8 J8

J2 J2, J10 J8 J8, C1 J1 J10 J1 J1, C1 J5, C1, C3 J5 J6, J11 J11 J5, J8 J1 J8 J8 e J4 J8 J8

J2 J2, J8 J8 J1 J1, J1 J8 J8 J5, J6, J1, J5, J2, e J8 e J4 J8 J4,

J2 J10 J8 J8 J1 J10 J1 J1, J8, C1 J2, J8, C1, C2 J2, J8, C1 J7, J11 J1 J2, J8, C4 J1, J8 J8 J8 e J4 J8 J8

J10

J10

J8 J11 J6 J8 J8

J8

J: C. jejuni, C: C. coli, the numbers show flaA genotypes.

profiles of flaA in C. jejuni and C. coli, respectively, with numeric designation (C. jejuni, 1e12; C. coli, 1e4). In 5 flocks, A, I, K, O, and S, 2 genotypes of C. jejuni or C. coli were found in the caecal contents from each of the tested flocks. In 8 flocks, A, D, E, G, I, M, O, and S, obtained from farm FA, 5 genotypes of C. jejuni (J1, J2, J5, J8, and J9) and 2 genotypes of C. coli (C1 and C2) were isolated from the caecal contents (Table 3). Among the first flocks slaughtered, almost all genotypes of Campylobacter from the chilled water, carcass, and/or chicken product samples were identical to those obtained from the caecal content samples originating from the same flock. For example, 3 genotypes (J5, J8, and C1) found in the caecal content samples from flock I were also present in the carcass, breast, tender, and liver samples from the same flock. Among the second flocks slaughtered, although some genotypes isolated from the carcass and/or chicken product samples were not identical to those obtained from the caecal contents from the same flock, these genotypes were identical to those found in the caecal content, chilled water, carcass, and/or chicken product samples originating from the first flock slaughtered on the same day. For instance, the J1 genotype observed in the carcass and chicken product samples from flock H was not present in the caecal content samples from the same flock, whereas the genotype was found in the caecal content, chilled water, carcass, and chicken product samples from flock G slaughtered on the same day. In addition, all 4 genotypes (J2, J5, J8, and C1) obtained from the carcass and chicken product samples originating from flock J, which was considered to be Campylobacter-negative, were present in the caecal content, carcass, and chicken product samples from flock I slaughtered on the same day. 4. Discussion It has been shown that the within-flock prevalence in Campylobacter-positive flocks is very high (Huneau-Salaün, Denis, Balaine, & Salvat, 2007; Jacobs-Reitsma, Bolder, & Mulder, 1994). Once a broiler is infected with Campylobacter spp., bird-to-bird transmission is extremely rapid and is enhanced by the coprophagic behaviour of chickens. As a result, the majority of birds in a positive flock become positive within only a few days (Shreeve, Toszeghy, Pattison, & Newell, 2000). Previously, it has also been reported that the concentrations of Campylobacter in caecal contents from Campylobacter-

infected birds are high and range from 4.1 to 10.6 log10 cfu/g (Berrang, Buhr, & Cason, 2000; Hue et al., 2011). In agreement with these previously published studies, our results show that high within-flock prevalence and high concentrations of Campylobacter in caecal contents from Campylobacter-infected birds is indeed associated with high prevalence of Campylobacter in carcasses, as well as breast and liver products, originating from these flocks. Some studies have suggested that defeathering and evisceration processes are the sources of carcass contamination with Campylobacter (Berrang et al., 2000; Figueroa, Troncoso, López, Rivas, & Toro, 2009; Guerin et al., 2010; Miwa, Takegahara, Terai, Kato, & Takeuchi, 2003; Ono & Yamamoto, 1999). In the present study, we observed that spray washing twice with slightly acidified chlorinated water after defeathering, evisceration, and soaking in chilled water containing FAC was not able to make carcasses free of Campylobacter at the abattoir investigated. In addition, the Campylobacter and TAB contamination levels in chilled water samples increased over time except 2 dates, 30 November and 7 December 2010; suggesting that chilled water that was contaminated with both Campylobacter and TAB from carcasses of the first flocks cross-contaminated the carcasses of the second flocks slaughtered on the same day. To prevent Campylobacter cross-contamination from chilled water, it is necessary to maintain the concentration of FAC in chilled water higher than 10 mg/L, higher concentration than those (1e5 mg/L) recommended by FSC. While flock Q was considered to be Campylobacter-negative based on the negative result of caecal content testing, Campylobacter was isolated from all the carcass samples but only after enrichment. Although the possibility of missampling cannot be excluded, we speculate that the feathers and skins of the birds might be contaminated with Campylobacter during transportation and waiting for slaughter in transport crates, as suggested previously (Newell et al., 2001). No Campylobacter was isolated from any chicken products of flock Q, suggesting chicken products originating from Campylobacter-negative flocks slaughtered prior to Campylobacter-positive flocks are free from Campylobacter crosscontamination. In addition, among the first flocks slaughtered, almost all genotypes of Campylobacter from the chilled water, carcass, and/or chicken product samples were identical to those obtained from the caecal content samples from the same flock. The results prove that the Campylobacter-negative flock did not

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introduce any Campylobacter into the abattoir, and the source of Campylobacter contamination was Campylobacter-positive flocks carried into the abattoir for slaughter and further processing. With regard to Campylobacter contamination in liver products, the rupture of intestines during the evisceration operation is not uncommon in abattoirs because the machinery used for evisceration is incapable of adjusting to natural variation in the size of broiler carcasses (Rosenquist, Sommer, Nielsen, & Christensen, 2006). In the present study, Campylobacter was isolated from not only all the liver products originating from Campylobacter-positive flocks but also all the liver products (5/5) from a Campylobacter-negative flock, which was the second flock slaughtered on 16 November 2010 and was cross-contaminated with Campylobacter. These results suggest that livers and the evisceration lines contaminated with Campylobacter, due to rupture of the intestines, led to the contamination of the cold water in the tank used for cooling livers. Once contaminated with Campylobacter, the evisceration lines and cold water in the tank subsequently contaminated livers originating from a successively slaughtered flock with Campylobacter. On the other hand, tenders, the underlying muscle of breast, are unlikely to be contaminated with Campylobacter from breast meat prior to cutting. However, tenders can be contaminated while being cut from the breast by equipment such as conveyer belts, cutting boards, knives, and gloves. In the present study, we show that the prevalence of Campylobacter in tender products from the second flocks slaughtered was significantly higher than that from the first flocks slaughtered on the same day. In addition, on 5 different slaughter dates, including 7 September, 19 October, 16 and 30 November, and 7 December 2010, flaA genotypes isolated from the first flocks slaughtered were found in tender products, but not the caecal contents, from the second flocks. These results show that processing lines of tenders contaminated with Campylobacter from processing tenders of Campylobacter-positive flocks subsequently contaminated tenders of the successively slaughtered flocks with Campylobacter. Together, our results show that when a Campylobacter-positive flock was slaughtered, the slaughter lines in an abattoir were contaminated with Campylobacter and that the concentration of Campylobacter in the processing line was sufficiently high to contaminate carcasses and chicken products of not only the flock but also a successively slaughtered flock with Campylobacter, even if the successively slaughtered flock was Campylobacter-negative. In this study, the Campylobacter concentrations in the products originating from the Campylobacter-negative flock were: close to the enumeration limit (1.7 log10 cfu/carcass) in the carcass samples; and below the enumeration limit (2.0 log10 cfu/g) in the liver samples. The mean Campylobacter concentrations in the carcasses and liver products originating from the 18 Campylobacter-positive flocks were 3.8 log10 cfu/carcass and 2.6 log10 cfu/g, respectively. Although the difference in Campylobacter concentrations would depend on slaughtering and processing procedures, Campylobacter concentration in chicken products originating from a Campylobacter-negative flock cross-contaminated by a Campylobacter-positive flock slaughtered immediately before would be lower than those in chicken products originating from Campylobacter-positive flocks as reported by other studies (Allen et al., 2007; Reich, Atanassova, Haunhorst, & Klein, 2008). Some quantitative risk assessments of Campylobacter in broiler meat have concluded that the most effective intervention measures are those which reduce the concentration of Campylobacter in carcasses (Lake, Hudson, Cressey, & Bayne, 2007; Rosenquist, Nielsen, Sommer, NØrrung, & Christensen, 2003). Specifically, Rosenquist et al. (2003) showed that the incidence of campylobacteriosis associated with chicken consumption was reduced 30 fold by introducing a 2 log reduction in the number of Campylobacter in the carcasses. In addition, while Campylobacter was present in only 1 of 5 breast products

originating from the Campylobacter cross-contaminated flock, the prevalence of Campylobacter in breast products from Campylobacter-positive flocks was very high (99%, 89/90), in accordance with the findings of Reich et al. (2008). Overall, our results suggest that the concentration of Campylobacter in carcasses is associated with the prevalence of Campylobacter in breast products. We recently found that Campylobacter was isolated from 67 of 142 (47%) flocks between September 2009 and February 2010 (Haruna et al., 2012). The farms investigated in this study adapted looser biosecurity measures than those studied in the previous study. Of 11 farms investigated in the present study, only 3 farms disinfected vehicles before entering, and only 4 farms exterminated rodents more than once every 3 months. In addition, all 8 flocks from farm FA, which did not implement the ‘all-in, all-out’ practice at the farm level, were found to be Campylobacter positive. These biosecurity measures may have an effect on Campylobacter colonization in broiler flocks. The reduction of Campylobacter prevalence in broiler flocks should be taken as an effective control measure for preventing introduction of Campylobacter into abattoirs and for consequently reducing Campylobacter prevalence in chicken products in addition to the good hygienic practice at abattoirs and logistic slaughter. The abattoirs slaughtering flocks with high Campylobacter prevalence like those in this study should encourage farmers to take strict biosecurity measures on their farms. Overall, the present study fills the data gap identified in the risk assessment reported by the FSC in 2009. Our results show that the prevalence and concentration of Campylobacter in chicken products derived from a Campylobacter-negative flock cross-contaminated by a Campylobacter-positive flock slaughtered immediately before were lower than those in chicken products originating from Campylobacter-positive flocks. In addition, the high prevalence of Campylobacter in broiler flocks to be slaughtered can result in the low probability of slaughtering a Campylobacter-negative flock immediately after a Campylobacter-positive flock. Conflict of interest The authors report no conflicts of interest. Acknowledgements The authors thank the staff at the abattoir for their kind cooperation. We also thank the JFRL staff for flaA typing. This study was funded by the Ministry of Agriculture, Forestry and Fisheries of Japan. References Allen, V. M., Bull, S. A., Corry, J. E., Domingue, G., Jorgensen, F., Frost, J. A., et al. (2007). Campylobacter spp. contamination of chicken carcasses during processing in relation to flock colonisation. International Journal of Food Microbiology, 113, 54e61. Berrang, M. E., Buhr, R. J., & Cason, J. A. (2000). Campylobacter recovery from external and internal organs of commercial broiler carcass prior to scalding. Poultry Science, 79, 286e290. EFSA Panel on Biological Hazards. (2011). Scientific opinion on Campylobacter in broiler meat production: control options and performance objectives and/or targets at different stages of the food chain. EFSA Journal, 9, 2105. Figueroa, G., Troncoso, M., López, C., Rivas, P., & Toro, M. (2009). Occurrence and enumeration of Campylobacter spp. during the processing of Chilean broilers. BMC Microbiology, 9, 94. Food Safety Commission (FSC) in Japan. (2009). Risk assessment of Campylobacter jejuni and Campylobacter coli in poultry. Evaluation report of microorganisms and viruses, 25-June (in Japanese). Guerin, M. T., Sir, C., Sargeant, J. M., Waddell, L., O’Connor, A. M., Wills, R. W., et al. (2010). The change in prevalence of Campylobacter on chicken carcasses during processing: a systematic review. Poultry Science, 89, 1070e1084. Haruna, M., Sasaki, Y., Murakami, M., Ikeda, A., Kusukawa, M., Tsujiyama, Y., et al. (2012). Prevalence and antimicrobial susceptibility of Campylobacter in broiler flocks in Japan. Zoonoses and Public Health, 59, 241e245.

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