High prevalence of pathogenic Yersinia enterocolitica in pig cheeks

Accepted Manuscript High prevalence of pathogenic Yersinia enterocolitica in pig cheeks Riikka Laukkanen-Ninios, Maria Fredriksson-Ahomaa, Riitta Maijala, Hannu Korkeala PII:

S0740-0020(14)00091-4

DOI:

10.1016/j.fm.2014.04.016

Reference:

YFMIC 2157

To appear in:

Food Microbiology

Received Date: 17 July 2012 Revised Date:

11 March 2014

Accepted Date: 27 April 2014

Please cite this article as: Laukkanen-Ninios, R., Fredriksson-Ahomaa, M., Maijala, R., Korkeala, H., High prevalence of pathogenic Yersinia enterocolitica in pig cheeks, Food Microbiology (2014), doi: 10.1016/j.fm.2014.04.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Pathogenic Y. enterocolitica in pork

High prevalence of pathogenic Yersinia enterocolitica in pig cheeks

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Riikka Laukkanen-Ninios1*, Maria Fredriksson-Ahomaa1, Riitta Maijala2, Hannu Korkeala1

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P.O. Box 66, FIN-00014 University of Helsinki, Finland

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Running title: Pathogenic Y. enterocolitica in pork

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Finnish Food Safety Authority Evira, Mustialankatu 3, FIN-00790 Helsinki, Finland

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Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine,

*Corresponding author

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Mailing address: Department of Food Hygiene and Environmental Health, Faculty of

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Veterinary Medicine, University of Helsinki, P.O. Box 66, FIN-00014 University of Helsinki,

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Finland

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Phone: +358 9 191 57135

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Fax: +358 9 191 57170

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E-mail address: [email protected]

Highlights

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More pathogenic Y. enterocolitica in pig cheeks than other pork products

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Pathogenic Y. enterocolitica detected in 39% of pork cuts using PCR

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Low numbers (0.1–1.6 MPN/g) of pathogenic Y. enterocolitica detected in pork

Cheeks are a possible Y. enterocolitica contamination and infection source

Quantification of pathogenic Y. enterocolitica in pork using MPN and PCR detection

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Pathogenic Y. enterocolitica in pork

Abstract

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Samples from pork cuts for minced meat and cheeks from processing plants and a

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slaughterhouse, and modified atmosphere (MA) packaged pork from retail were studied to

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estimate the prevalence of pathogenic, i.e. virulence plasmid bearing, Yersinia enterocolitica

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and Yersinia pseudotuberculosis in pork, as well as to quantify pathogenic Y. enterocolitica in

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pork cuts. Pathogenic (virF-positive) Y. enterocolitica was isolated from 17 pig cheeks (23%)

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but not from any of the MA-packaged 54 retail pork samples and only from one of the 155

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pork cut (0.6%). Most (16/17) of the cheek samples were contaminated with pathogenic Y.

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enterocolitica 4/O:3 and one with bioserotype 2/O:9. No Y. pseudotuberculosis was isolated.

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The prevalence of pathogenic Y. enterocolitica was clearly higher (39%) in 155 pork cuts

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when studied with nested PCR targeting yadA on the virulence plasmid pYV although the

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contamination level was low varying between 0.1 and 1.6 MPN/g. Raw pork cuts and

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especially pig cheeks may serve as possible sources for yersiniosis caused by pathogenic Y.

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enterocolitica.

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Keywords: Yersinia enterocolitica, pork, musculus masseter, zoonosis

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Pathogenic Y. enterocolitica in pork

Introduction

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Yersinia enterocolitica and Yersinia pseudotuberculosis are zoonotic bacteria causing

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yersiniosis, a frequently reported bacterial zoonosis in the European Union (EFSA and ECDC

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2013). Pathogenic, i.e. virulence plasmid (pYV) bearing Y. enterocolitica is frequently

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isolated from pigs and also from pig carcasses (Laukkanen et al. 2010) providing a

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contamination source for pork. Pathogenic Y. enterocolitica is also isolated from pork

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products, but less frequently. The prevalence of pathogenic Y. enterocolitica in raw pork has

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been only 0–5% using isolation, but 5–36% using PCR method in the same samples

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(Boyapalle et al. 2001, Fredriksson-Ahomaa and Korkeala 2003, Thisted Lambertz et al.

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2007, Messelhäusser et al. 2011, Lucero Estrada et al. 2012). The prevalence of pathogenic Y.

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enterocolitica can also be affected by the meat selection. In a recent study, the detected

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prevalence of Y. enterocolitica O:3 in retail pork was 1%, 8% and 26% for retail minced pork,

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cheeks, and tongues, respectively, using isolation method (Messelhäusser et al. 2011). pYV-

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bearing Y. pseudotuberculosis can also be detected from pigs and pig carcasses, but less

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frequently than Y. enterocolitica (Laukkanen et al. 2010) and Y. pseudotuberculosis is only

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rarely isolated from pork (Fukushima 1985, Fukushima et al. 1997).

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Information on the prevalence of pathogenic Y. enterocolitica in pork in addition to the

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quantity in positive products is needed to further understand the epidemiological routes and

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relevant sources of yersiniosis as well as to estimate the exposure in quantitative microbial

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risk assessment. However, there is only little quantitative data available; the data is from a

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limited number of studies with a small number (N=19–28) of pathogenic Y. enterocolitica-

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positive retail samples, quantified with most-probable number (MPN) technique using serial

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dilution with multiplex PCR or traditional plating detection (Hudson et al. 2008, Bonardi et

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al. 2010, Messelhäusser et al. 2011).

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Pathogenic Y. enterocolitica in pork

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The aim of this study was to investigate the prevalence of pathogenic Y. enterocolitica and Y.

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pseudotuberculosis in raw pork and to estimate the quantity of pathogenic Y. enterocolitica in

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pork cuts.

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5 1. Materials and methods

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1.1 Sampling

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Altogether 281 pork samples were collected in order to study the prevalence of pathogenic Y.

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enterocolitica and Y. pseudotuberculosis in pork. Cheek samples (74) were collected from a

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slaughterhouse and a meat processing plant. Additionally 52 modified atmosphere (MA)

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packaged raw pork samples from various manufacturers were collected from six different

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retail stores. All retail samples were stored in 4°C and studied prior best before date.

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A total of 155 samples from pork cuts for minced meat were collected from crates during

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meat cutting (before mincing) at four meat cutting plants. In addition to cultivation, these

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samples were used to detect and quantify pathogenic Y. enterocolitica with nested PCR.

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Sampling was done in several rounds with 5 to 20 samples collected at one sampling time.

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1.2 Isolation and determination of Yersinia enterocolitica and Yersinia pseudotuberculosis

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All 281 pork samples were examined for Y. enterocolitica and Y. pseudotuberculosis using

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cold enrichment in peptone-mannitol-bile salts (PMB) broth and selective enrichment in

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irgasan-ticarcillin-potassium chlorate (ITC, prepared according to ISO 10273) broth for Y.

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enterocolitica except 24 cheek samples, which were studied only with cold enrichment. In

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brief, 10 g of pork was cut and diluted in 90 ml of PMB and mixed thoroughly. A volume of

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1ml of PMB was inoculated in 9 ml of ITC broth and incubated at 25°C for 48 h. PMB broth

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was cold enriched at 4°C for 7 and 14 days. Alkali treatment (0.5 ml of the sample was mixed

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with 4.5 ml of 0.25% KOH solution for 20 s before cultivation) was used after 14 days of cold

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enrichment in PMB. 100 µl of sample was streaked after each enrichment step onto

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cefsulodin-irgasan-novobiocin agar plate (Yersinia Selective Agar Base [CIN], Oxoid,

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Basingstoke, UK) and incubated at 30°C for 18 to 20 h and then further at 22°C for 24 h. One

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to three suspect colonies were streaked onto tryptic soy agar (TSA, Difco, Maryland, USA)

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plates for pure culture. Isolates were tested for urea hydrolysis using urea agar slants and

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isolates hydrolysing urea were identified using API 20E (BioMérieux, Marcy l’Etoile, France)

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according to manufacturer’s instructions with the exception of incubation at 25°C for 18 to 20

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h. Pathogenic Y. enterocolitica was further identified using PCR targeting chromosomal

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virulence gene ail and virF gene in the pYV according to Nakajima et al. (1992). Y.

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enterocolitica isolates were biotyped according to the revised scheme of Wauters et al. (1987)

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and serotyped with slide agglutination using commercial polyvalent O:1 and O:2, O:3, O:5,

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O:8 and O:9 antisera (Denka Seiken, Tokyo, Japan).

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1.3 Detection and quantification of yadA-positive Y. enterocolitica with nested PCR

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In order to estimate the number of pathogenic Y. enterocolitica in 155 pork cuts for minced

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meat from the meat cutting plants, MPN method of 4 fold dilution (1g–0.001g) with three

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replications was used for the first 2 batches (20 samples). However, due to the very low

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contamination level, MPN of 10 parallel 1 g subsamples from each sample was used as

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described earlier (Lindström et al. 2001) for the remaining 135 samples. Samples were

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analyzed after overnight enrichment in tryptic soy broth (TSB, Difco, Maryland, USA) with

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nested PCR targeting the yadA gene in the pYV (Kapperud et al. 1993) with modifications

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described by Fredriksson-Ahomaa et al. (Fredriksson-Ahomaa et al. 1999).

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1.4 Statistical analyses

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Confidence intervals for the detected prevalence using isolation method for 281 samples and

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X2 test for the comparison of isolation and nested PCR methods in 135 pork cut samples were

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calculated with the assumption of random sampling and using Epi Info 6.04d Epitable and

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Statcalc calculators (CDC, USA).

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6 2. Results

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Pathogenic, (virF-positive), Y. enterocolitica 4/O:3 was isolated from 16 (22%) and

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pathogenic Y. enterocolitica 2/O:9 from one pig cheek sample (5%) (Table 1). Pathogenic Y.

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enterocolitica 4/O:3 was isolated from one pork cut sample for minced meat (0.6%) but not

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from retail pork samples. The isolation rate of pathogenic Y. enterocolitica in cheeks (23%)

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was significantly higher than that of retail pork products (0%) or pork cuts for minced meat

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(0.6%) (Χ2, P<0.001). No Y. pseudotuberculosis was isolated from pork samples in this study.

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Two cheek samples harbored Y. enterocolitica 4/O:3 isolates carrying chromosomal virulence

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gene ail and virF in pYV as well as isolates carrying ail but not virF, and four of the pig

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cheeks harbored only ail-positive but virF-negative Y. enterocolitica 4/O:3 isolates.

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Using nested PCR targeting the yadA in the pYV, pathogenic Y. enterocolitica was detected in

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60 of 155 (39%) meat cut samples and the detected prevalence varied from 10% to 57%

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between four meat cutting plants (Table 2). The detection rate of nested PCR using 10

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subsamples in 135 samples was significantly higher than that of isolation method (Χ2,

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P<0.001) for the same samples. The MPN estimate of the 60 PCR-positive samples ranged

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from 0.1 to 1.6 per 1 g of pork.

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3. Discussion

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Pathogenic Y. enterocolitica was isolated frequently (23%) and significantly more often from

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cheeks than from retail pork samples (0%) or from pork cuts for minced meat (0.6%)

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indicating that pig cheeks are heavily contaminated with pathogenic Y. enterocolitica. In

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addition, it has been shown that Y. enterocolitica 4/O:3 can grow well on raw cheek meat

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packaged in MA at 6°C even in the presence of high number of lactic acid bacteria

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(Fredriksson-Ahomaa et al. 2012) emphasizing pork, cheek meat in particular, as an important

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source of pathogenic Y. enterocolitica. Previously, Y. enterocolitica has been isolated from

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8–36% of pig cheeks (Wauters et al. 1988, Shiozawa et al. 1991, Messelhäusser et al. 2011)

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and the viable cell count in fresh cheeks has been from <10 cfu/g (Fredriksson-Ahomaa et al.

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2012) up to 3.0x102–2.7x103 cfu/g (Shiozawa et al. 1991) when cheeks were studied with

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serial dilution and plate count. Cheeks are often used in heated meat products, but they are

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also served as home prepared food as well as a delicacy in restaurants. Cheeks are also used in

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brawn that has been associated with yersiniosis outbreaks (Marjai et al. 1987, Grahek-Ogden

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et al. 2007). The high prevalence with high quantity of pathogenic Y. enterocolitica in cheeks

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emphasizes the need for good hygiene practices in meat processing plants to avoid

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contamination of equipment and products as well as vigilance at homes and restaurants for

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proper cooking times and prevention of cross-contamination.

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Since all MA-packaged retail pork samples and all but one of the pork cut samples for minced

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meat were negative for pathogenic Y. enterocolitica using culturing, we estimated the

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prevalence and quantity of pathogenic Y. enterocolitica in the pork cut samples for minced

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meat using sensitive nested PCR method targeting yadA in pYV. Altogether 39% of the 155

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samples proved to be positive, and the isolation rate (0.6%) was significantly lower.

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Pathogenic Y. enterocolitica in pork

The quantity of pathogenic Y. enterocolitica in pork cuts at the meat cutting plants varied

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between 0.1–1.6 MPN/g, indicating a low contamination level in the samples. No previous

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estimations on the contamination levels of pork cuts at meat processing plants were reported

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in the literature to our knowledge. On retail level, Bonardi et al. (2010) studied 19 raw pork

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samples using isolation after 3-tube serial dilution and detected ail-positive Y. enterocolitica

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2/O:9 from one sample with the MPN contamination level of 0.92/g in the sample. The level

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of contamination of 26 steaks and schnitzels in retail was 0.30–5.42 MPN/cm2 using PCR

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detection with 3-tube MPN (Hudson et al. 2008). The contamination level appears to be low

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in pork products other than cheeks, where 23% positive samples with just isolation method

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suggests high quantity of pathogenic Y. enterocolitica in the samples.

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The prevalence with nested PCR method of pathogenic Y. enterocolitica-positive pork cuts

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for minced meat varied considerably (10–57%) between the four meat cutting plants. This

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variation can be affected by hygiene practices applied and the cross contamination at the meat

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cutting (Fredriksson-Ahomaa et al. 2004) as well as the contamination level of the carcasses

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entering meat cutting plants which in its turn depends on the prevalence of Y. enterocolitica in

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pigs and slaughter hygiene (Laukkanen et al. 2009, Laukkanen et al. 2010). Over half of the

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fattening pigs in Finland carry pathogenic Y. enterocolitica (Korte et al. 2004, Laukkanen et

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al. 2010) presenting a high contamination pressure for carcasses and subsequently pork.

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Based on this study, raw pork cuts and especially pig cheeks may serve as possible sources

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for yersiniosis caused by pathogenic Y. enterocolitica.

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Acknowledgements

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This study was supported by research funding from Kyllikki and Uolevi Lehikoinen

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foundation and the Ministry of Agriculture and Forestry, Finland (4877/501/2005), and was

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Pathogenic Y. enterocolitica in pork

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performed at the Centre of Excellence in Microbial Food Safety Research, Academy of

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Finland (118602, 141140).

3 We thank Jari Aho, Erja Merivirta, and Anu Seppänen for their technical support. Anne

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Paloranta is kindly acknowledged for her help in sampling as well as processing plants for

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their co-operation.

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7 References

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Tables

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Table 1. Isolation of Yersinia enterocolitica from pork samples Na

Sample Pork cuts for minced meat Retail pork samples Pig cheeks Total

155 52 74 281

Pos. 1 0 17b 18

virF-positive samples Prev. (%) CI of prev. 0.6 0.03–4.08 0 0.00–8.57 23 14.32–34.48 6 3.95–10.11

Pathogenic Y. enterocolitica in pork

Pos. 1 0 21 22

ail-positive samples Prev. (%) CI of prev. 0.6 0.03–4.08 0 0.00–8.57 28 18.80–40.23 8 5.08–11.78

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isolates of bioserotype 2/O:9 was identified. In addition, two samples harbored both virF-

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positive and virF-negative Y. enterocolitica

CI, confidence interval; N, Number of samples; Pos., positive; Prev., prevalence

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In 16 samples, all pathogenic isolates belonged to bioserotype 4/O:3 and in one, only

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Table 2. Detection and quantification of yadA-positive Yersinia enterocolitica in pork samples Sampling round 1 2 3 Total

N 10b 5 5 20

Positive samples (%) 1 (10)c 0 (0) 1 (10) 2 (10)

1 2 3 4 5 6 Total

10b 10 10 10 15 20 75

0 (0) 1 (10) 0 (0) 8 (80) 6 (40) 19 (95) 34 (45)

III

1 2 Total

15 15 30

5 (33) 2 (13) 7 (23)

IV

1 2 Total

15 15 30 155

Total

0.22/g

1.00; 0.11–1.61/g 0.26; 0.11–0.51/g 0.56; 0.11–1.61/g 0.60; 0.11–1.61/g

a

9 (60) 8 (53) 17 (57) 60 (39)

0.11; 0.11–0.11/g 0.17; 0.11–0.22/g 0.13; 0.11–0.22/g 0.21; 0.11–0.36/g 0.11; 0.11–0.11/g 0.16; 0.11–0.36/g 0.41; 0.11–1.61/g

Average; minimum–maximum values of most probable number (MPN) per positive sample Samples were studied after serial dilutions instead 10 parallel 1 g samples c One culture positive sample

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0.22/g 0.31; 0.22–0.4/g

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MPN of positive samplesa 0.4/g

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Meat cutting plant I

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