Prevalence of major foodborne pathogens in food confiscated from air passenger luggage.

FOOD-06628; No of Pages 10 International Journal of Food Microbiology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Prevalence of major foodborne pathogens in food confiscated from air passenger luggage Dagmar Schoder a,b,⁎,1, Anja Strauß a,1, Kati Szakmary-Brändle a, Beatrix Stessl a, Sabine Schlager c, Martin Wagner a a b c

Institute for Milk Hygiene, Milk Technology and Food Science, University for Veterinary Medicine, Vienna, Austria Veterinarians Without Borders, Austria Austrian Agency for Health and Food Safety, Graz, Austria

a r t i c l e

i n f o

Available online xxxx Keywords: Listeria monocytogenes Coagulase positive staphylococci Verotoxigenic Escherichia coli (VTEC) Salmonella spp. Bush meat Airport

a b s t r a c t The EU has issued several directives and regulations pertaining to the importation of animals and products of animal origin (POAO) and veterinary controls on importation. Unfortunately, little information is available concerning associated risks and no attempts have been made to collect baseline data on the actual prevalence of zoonotic agents in POAO carried by travellers. To meet these challenges the EU recently introduced and financed a research project “PROMISE”. Its main objectives were to assess the risks involved when foodborne pathogens are introduced to the EU via uncontrolled imports. With special permission of the Austrian health authorities, spot-checks were made of the luggage of 61,355 passengers from 240 flights from non-EU countries arriving at the Vienna International Airport (VIE airport). Over a period of eight months (August 2012 through March 2013) 1473 POAO items were confiscated. A total of 600 samples were suitable for Salmonella spp., Campylobacter spp., verotoxigenic Escherichia coli and Listeria monocytogenes prevalence analysis. Foodborne pathogens could be detected in 5% (30/600) of all samples. The highest prevalence was attributed to L. monocytogenes, at 2.5%, followed by VTEC and Salmonella spp. at 1.3% and 1.2%, respectively. Campylobacter spp. was not present in any of the 600 samples. Multi-locus sequence typing (MLST) of L. monocytogenes revealed that current sequence types (ST) corresponded to the worldwide most present clonal complexes 1, 2, 3, 5, 9, and 121. Generally, L. monocytogenes ST9 was the predominant allelic profile, which was mainly isolated from Turkish meat products. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The objective of the European Union (EU) food safety policy is to protect consumer health. To achieve this objective the EU ensures that control standards for food and food hygiene, and animal health and welfare are established and implemented. However, this must be balanced by other ambitions. For example, the 1957 Treaty of Rome and its revisions (EuroKnow, 2012) stipulate freedom of movement of goods, people and services within the EU. This not only has an economic foundation, but also serves general public interests. Since the EU Schengen Agreement was established in 1985, and further expanded in 1997, there has been even more freedom of travel for people and trading of goods, including food products. In the context of freedom of movement of goods this has pushed controls to the outer borders of the EU. As a consequence the limited numbers of EU border inspection ⁎ Corresponding author. Tel.: +43 1 25077 3520; fax: +43 1 25077 3590. E-mail address: [email protected] (D. Schoder). 1 Joint first authors.

posts focus mainly on travellers and commercial imports from third countries, which are those countries that are not members of the EU. These posts are found at airports, sea ports and major railway, road and post entry stations. The EU has issued several directives and regulations pertaining to the importation of animals and products of animal origin (POAO) and veterinary controls on importation: EC 206/2009 (covering POAO), EC 97/78, 91/496/EC, EC 136/2004 and EC 2007/275 (veterinary checks on animals and POAO at border inspection posts; EC, 1991, 1997, 2004, 2007, 2009a). However, most of these fundamentals only refer to commercial trade in food and food products and usually only in large quantities (EC, 2012). Nonetheless, some food products are exempted from customs control. Among these products are those intended for personal consumption, which are potentially present in traveller luggage, those sent by post in small volumes to individuals and those sent as trade samples (EC, 2012; FVO, 2013; see also directives 91/496/EC and 97/78/EC (EC, 1991, 1997)). Transmission of zoonotic bacteria to humans can either occur via animal contacts or by contaminated food. Unfortunately, little information

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.08.010 0168-1605/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Schoder, D., et al., Prevalence of major foodborne pathogens in food confiscated from air passenger luggage, Int. J. Food Microbiol. (2014), http://dx.doi.org/10.1016/j.ijfoodmicro.2014.08.010

2

D. Schoder et al. / International Journal of Food Microbiology xxx (2014) xxx–xxx

is available concerning associated risks and no attempts have been made to collect baseline data on the actual prevalence of zoonotic agents in POAO carried by travellers. To meet these challenges the EU recently introduced and financed a research project, “PROMISE” (protection of consumers by microbial risk mitigation through combating segregation of expertise) (Promise, 2012). PROMISE is coordinated by the University of Veterinary Medicine, Vienna, Austria. The project's main objectives are to assess the risks involved when foodborne pathogens are introduced to the EU via uncontrolled imports. With special permission of the Austrian health authorities, spot-checks on the luggage of passengers from 240 flights from non-EU countries arriving at the Vienna International Airport (VIE airport) were inspected over a period of eight months (August 2012 through March 2013). When POAO were detected, they were confiscated and subsequently analysed microbiologically for the prevalence of a range of foodborne bacterial pathogens, including Salmonella spp., Campylobacter spp., verotoxigenic Escherichia coli, coagulase-positive staphylococci (SA) and Listeria monocytogenes. 2. Material and methods 2.1. VIE airport: customs applicable flights VIE airport acts as a new border to EU-third countries (non-EU members) and connects the EU closely with Northern Asia (Turkey, Russia), the Middle East (Arab countries) and the Far East (China). The airport includes a food and veterinary inspection service that covers non-EU and non-Schengen countries. In 2012 the total number of passengers (transfer, departure and arrival) passing through VIE airport was 22,165,794 and the total number of flights serviced was 244,650. More than 58,000 flights were categorized as customs applicable flights. Customs applicable flights are those flights conveying passengers from EU-third countries and flights from EU countries conveying transit passengers. 2.2. Checks by Austrian customs on traveller luggage for POAO The EU has issued several directives and regulations concerning importation of foods and POAO: Commission Regulation No 206/2009 (EC, 2009a) states that all animal products originating from animals from third countries must be subjected to veterinary inspection. Among these products are those intended for personal consumption (potentially present in travellers' luggage), those sent by post in small volumes to individuals and those sent as trade samples corresponding to 91/496/EC and 97/78/EC (EC, 1991, 1997). However, as comprehensive veterinary inspection is not possible, importation of a POAO is generally prohibited. As a result customs officers at VIE airport are obligated to perform spot-checks on arrival passengers. In contrast, air passengers within the EU are permitted to carry POAO for personal consumption. These spot-checks are performed on the basis of individual risk analysis (type of luggage, origin and behaviour of travellers) and on the basis of ongoing animal disease reports and country risk assessments. On account of animal disease reports from the World Organization for Animal Health (OIE) and the Food and Agriculture Organisation of the United Nations (FAO), customs applicable flights are routinely categorized as either high or low risk flights. During the investigation period flights departing from Turkey, Egypt, China, the United Arab Emirates, Russia and countries of the former Soviet Union were classed as high risk flights. Customs officers were instructed to increase inspections of passengers from these high risk flights. In the context of seizures customs officers retained information regarding the passenger flight data, flight number (data not shown), country of origin, airport of departure, whether or not there was a flight stop-over and the amount of sample material seized (total weight and number of pieces) for each POAO.

2.3. Austrian Ministry of Health exceptional approval of customs-seized POAO investigation On the 14th of May, 2012 the University of Veterinary Medicine in Vienna (VUW) received essential authorization by the Austrian Ministry of Health, Department B/10 for Veterinary Law, Animal Health and Trade with Living Animals, to examine seized POAO present in the luggage of travellers from outside the EU for scientific purposes. To comply with the authorization the following terms and conditions were established for the practical procedure at the border inspection point at VIE airport and the destination laboratory: (i) VUW, in cooperation with the Austrian Agency for Health and Food Safety (AGES), had to complete a detailed procedure statement about POAO sample collection and pickup at the VIE airport border inspection point. (ii) VUW was obliged to issue an accompanying document for each sample shipment, stating the following: Country of origin or flight number of the traveller from which the consignment was taken. This included the scientific name of the seized material, the animal species from which the material originated and a description to allocate the consignment to the document and the precise destination in Austria. (iii) Exact instructions in the accompanying document as to how to proceed in the event of incidents during transport to the delivery address, the name of the responsible contact person and how decontamination should be performed, including the substance to be used. (iv) Each movement was to be accompanied by an appropriate accompanying document. (v) Samples had to be transported in sealed impervious containers. Packaging had to comprise at least three layers: two layers had to be impermeable, pressure-resistant and shock-proof and the container had to be surrounded by absorbent material. (vi) Each container had to be marked in accordance with international regulations (UN 3373, UN 2814 or UN 2900). Containers or their external packaging had to be clearly marked “only for use in the laboratory” or “for laboratory use only.” (vii) Samples had to be sent from the border inspection post at VIE airport directly to the Austrian L3 High Security Laboratory. (viii) Sample materials had to undergo risk assessment at AGES, including all necessary investigations. (ix) Packaging and non-essential research material had be incinerated in a disease-proof manner using an approved disposal system for clinical or laboratory waste, according to European Regulation No. 1069/2009 (EC, 2009b). (x) All required work had to be conducted in a manner to secure and exclude danger to Austrian livestock. 2.4. POAO sample collection and pickup at VIE airport border inspection post Six different categories of POAO samples could be differentiated: (i) milk and all types of milk products, including milk powder and baby-food containing milk; (ii) eggs and egg products, including egg powders; (iii) honey; (iv) meat and meat products (v) fish and marine products and (vi) bush meat. Bush meat is a specific designation for animal game hunted in the jungle or savannah, most usually in Africa. This particularly includes not only meat from duiker (small antelope), rats, apes and porcupines, but also meat from other mammals (such as elephants, buffalos), birds, reptiles and amphibians. From all POAO confiscated by customs, 600 samples were selected for further study. The following selection criteria were implemented: samples had to have a minimum weight of 300 g. Raw, ready-to-eat, perishable and exotic POAO (e.g. bush meat) were given preference. All 600 samples selected for the study were classified into three categories: (i) ready to eat, (ii) raw products anticipated to require preparation, clearly not in edible form, and (iii) canned goods, sealed with tin-plate or aluminium.



2.5. Sample transfer and transport to the laboratory Samples were acquired directly or stored at 4 °C in a designated refrigerator until the following day at the border inspection post at VIE airport. Transfer of sample material was performed under terms of the Austrian Federal Ministry of Health, as described above. Samples were transported in sealed, epidemic-safe aluminium crates (Hobbock-Safety boxes, accredited by the European Agreement Concerning the International Carriage of Dangerous Goods by Road (ADR), 513016 (Dosenprofi, Kufstein, Austria), declared with “UN 3373 — Biological Substance Category B” and “for laboratory use only” labels in accordance with the “Guidance on Regulations for the Transport of Infectious Substances, 2011–2012” (WHO, 2010). Crates were transported by car to the Austrian L3 High Security Laboratory of the AGES in Vienna. Packaging and non-essential research materials were incinerated in a disease-proof manner, in accordance with European Regulation No. 1069/2009 (EC, 2009b). 2.6. Pooling of samples An approximately 1 kg representative sample was taken from the total quantity of an individual food item seized from each passenger. Passengers often carried more than 1 kg of a particular POAO. This procured sample comprised all components of that particular item (margins, centre, breadcrumb coating, egg shells, skin, etc), which were prepared using a sterile knife. This 1 kg sample was chopped, mixed and homogenized using sterile equipment. Subsequently a 300 g aliquot of homogenate was removed for microbiological investigation. Thoroughly mixed samples were weighed into stomacher bags for the various assays. 2.7. Microbiological and molecular biological investigations POAO samples were analysed for prevalences of the major foodborne pathogens: L. monocytogenes (ISO, 2004), Campylobacter spp. (ISO, 2006), Salmonella spp. (ISO, 2002), verotoxigenic E. coli (ISO, 2012) and coagulase-positive staphylococci (ISO, 1999a,b). Additionally, total bacterial (ISO, 2003) and E. coli (ISO, 2001) counts were determined for each sample. 2.7.1. L. monocytogenes 25 g samples to be investigated were added to 225 ml Half Fraser (HF) bouillon (Biokar Diagnostics, Beauvais, France), mixed in a stomacher bag and homogenized with a stomacher blender at a power and duration dependent upon the texture of the food. HF bouillon broths were incubated at 30 °C for 24 h. Subsequently, 100 μl aliquots of HF pre-enrichments were transferred to 10 ml of Full Fraser (FF) (Biokar) enrichment broth, followed by incubation at 37 °C for 48 h, according to ISO 11290 (1996). Aliquots from HF and FF broths were streaked onto both Aloa (Merck, Darmstadt, Germany) and Palcam (Biokar) agars. Plates were incubated at 37 °C for 48 h and inspected for characteristic Listeria-suspicious colonies. Plates were rinsed with 1 ml of 0.01 M Tris–buffer and DNA isolated by the quick isolation method with Chelex, described by Walsh et al. (1991). DNA was analysed by PCR for the presence of the iap (Bubert et al., 1999) and 16SrRNA/hly (Border et al., 1990) genes following electrophoresis of fragments in a 1.5% agarose gel containing 0.5× TBE Tris–Borate electrophoresis buffer (TBE) and 3.5 μl SYBR Safe DNA gel stain (Invitrogen, Eugene, Oregon, USA), at 120 V for 25 min. GeneRuler 100 bp (MBI Fermentas, St. Leon-Rot, Germany) was used as a standard. Fifteen L. monocytogenes isolates were characterized by MLST typing based on the following seven housekeeping loci: abcZ (ABC transporter), bglA (beta glucosidase), cat (catalase), dapE (succinyl diaminopimelate desuccinylase), dat (D-amino acid aminotransferase), ldh (L-lactate dehydrogenase) and lhkA (histidine kinase), according to Ragon et al. (2008). An allele number was assigned to each housekeeping gene. Subsequently,

3

sequence types (ST) were determined and compared using the Institute Pasteur L. monocytogenes MLST database (http://www.pasteur.fr/ recherche/genopole/PF8/mlst/Lmono.html accessed: 25.01.2014). An allelic profile-based comparison, applying a minimum spanning tree (MST), was performed to define the relationships among strains at the microevolutionary level (MSTree plugin, Institute Pasteur). 2.7.2. Salmonella spp. 25 g samples to be investigated were added to 225 ml of buffered peptone water (Oxoid, Basingstoke, Great Britain), mixed in a stomacher bag and homogenized with a stomacher blender at power and duration dependent upon the texture of the food. This reconstitution step was performed at 37 °C for 18 h. Enrichment steps were carried out with two selective bouillons. Rappaport-Vassiliadis medium with soya (Oxoid) was inoculated with 100 μl of the reconstituted medium and incubated at 42 °C for 24 h. Mueller–Kauffmann tetrathionate/novobiocin plus iodine medium (Oxoid) was inoculated with 1 ml of reconstituted medium and incubated at 37 °C for 24 h. Enrichment broths were streaked onto xylose–lysine–desoxycholate agar (XLD, Merck) and chromogenic SM ID2 agar (bioMérieux, Marcy lÈtoile, France). Plates were incubated at 37 °C for 24 h and inspected for characteristic Salmonella colonies. Colonies were rinsed and confirmed by PCR (Rahn et al., 1992). 2.7.3. Campylobacter spp. Bolton broth, containing the Bolton antibiotic supplement and 5% lysed horse blood (Oxoid), was used as culture medium for Campylobacter spp. identification. Briefly, 25 g of food sample was added to 225 ml of Bolton broth, homogenized by a stomacher blender and incubated for 48 h at 42 °C. The enrichment was streaked onto modified charcoal cefoperazone deoxycholate agar (mCCDA, Oxoid) and CampyFood Agar (bioMérieux). Plates were incubated at 42 °C and examined after 48 h. Enriched broth and plates were incubated in a micro-aerophilic atmosphere that was generated in an anaerobic jar using the CampyGen gas-generating system (bioMérieux). All colonies suspicious of Campylobacter were checked by determining the presence of oxidase activity, microscopic appearance (motility, spirals) and confirmed by PCR (Wang et al., 2002). 2.7.4. Coagulase positive staphylococci (SA), E. coli and total bacterial count at 30 °C 10 g samples were homogenized with 90 ml of peptone water (Oxoid) in a stomacher blender. Decimal dilution lines were prepared from the initial suspension with Ringer solution down to 10−8. 100 μl of the initial suspensions and the decimal dilutions were plated onto BP-RPF agar (Oxoid) in duplicate. Plates were incubated for 48 h at 37 °C and inspected for characteristic SA-colonies. For E. coli 1 ml aliquots from the initial suspension and further dilutions were poured with melted chromID-Coli agar (BioMérieux) in duplicate. After 24 h of incubation at 37 °C plates were inspected for the presence of characteristic pink colonies, which were counted. For total bacterial counts 1 ml of the dilutions were poured with standard plate count (PCA) agar (Oxoid). Plates were inspected for colonies and counted after 72 h of incubation at 30 °C. 2.7.5. Verotoxigenic E. coli 25 g samples were homogenized with 225 ml of modified CASObouillon plus novobiocin (BO0869S, Oxoid) in a stomacher blender. Incubation was performed at 42 °C for 20 h; during which 1.6 ml aliquots were removed after 6 h and after 20 h, mixed with 400 μl of glycerol (60%) and frozen at −80 °C until further investigation. 100 μl aliquots of the thawed 20 h enrichment-bouillons were transferred into 4 ml of CASO bouillon (CM0129B, Oxoid) containing novobiocin 10 mg/l (SR0181E) and mitomycin C, 50 μg/l (A2190.0002, AppliChem, Darmstadt, Germany), incubated at 37 °C for 16–18 h and


4


screened for verotoxins 1 and 2 by enzyme-linked immunoassay Ridascreen Verotoxin (R-Biopharm, Heidelberg, Germany). For confirmation of toxin genes and serotype identification verotoxin-positive samples in Ridascreen assay (n = 8) were sent to the Austrian national reference laboratory for E. coli (AGES) as deep frozen (dry ice) enrichment broths (20 h enrichments) together with the mitomycin C-enriched broths. For further investigations enrichment broths were tested for the presence of genes coding for stx1, stx2 and eaeA. Isolation of VTEC strains was performed according to a standard protocol (ISO/TS, 13136, 2012). The strains were confirmed as E. coli using VITEK 2® (bioMérieux). Sorbitol fermentation was determined with sorbitol McConkey agar (Oxoid), beta-glucuronidase activity with CHROMagar™ O157 (Becton Dickinson, New Jersey, USA), and the enterohemolysis phenotype on enterohemolysin agar (Heipha, Eppelheim, Germany). Isolate virulence genes (stx1, stx2, Ehly and eaeA) were confirmed using two duplex PCRs (Reischl et al., 2002). 3. Results 3.1. Total number of confiscated POAO During the investigation period between August 2012 and March 2013 a total of 78,415 flights landed at VIE airport, 75% (58,795) of which were categorized as customs applicable flights, which carried 5,385,000 passengers (http://www.flightstats.com). A total of 61,355 passengers from customs applicable flights were inspected on the criteria of an individual risk analysis and country risk assessments based on animal disease reports. Of these inspections 1473 POAO

were established in passenger luggage. The weight of all confiscations totalled 6229 kg. 86.4% of confiscated POAO originated from airports of countries considered at high risk of communicable animal diseases. The breakdown was as follows: 58.5% of POAO came from Turkey (3642 kg), 14.0% from Egypt (871.5 kg), 6.4% from China (398.5 kg), 3.3% from the United Arab Emirates (208.5 kg) and 4.2% from Russia (including the former Soviet Union; 259.5 kg). The remaining 13.6% (849 kg) came from other countries (Karner, 2012, personal communication). 3.2. POAO selected for further analysis All selected POAO (n = 600) were established in passenger luggage from 240 different flights. Individual samples were between 300 g and 15 kg in weight. Total weight of the 600 confiscated samples was 1278 kg. A breakdown of records revealed that 81.1% and 13.5% of samples originated from Asia and Africa, respectively. The remaining 5.2% came from European countries not in the EU. A single sample was traced to Brazil (Table 1). More than half of samples (51.8%) originated from Turkey. These 311 samples were obtained from spot-checks from passengers from 121 direct flights from four Turkish airports (Istanbul, Ankara, Antalya and Izmir). 3.3. Packaging 38.7% of all POAO samples (232/600) were in their original packaging, whereas the remaining 61.3% were packed by the respective passengers. A total of 12 package materials could be differentiated. 23.5% and 44.8% of

Table 1 Total number and origin of confiscated POAO.

a a a a a a a b b b b b b b b b b b b b b b b b b b c c c c c c c d

a b c d

Country of departure

Samples/country

% of total

Côte d'Ivoire Egypt Ethiopia Mozambique Nigeria South Africa Tunisia Armenia Azerbaijan China India Iran Israel Jordan Kazakhstan Korea Mongolia North Korea Philippines Qatar Russia South Korea Thailand Turkey United Arab Emirates Vietnam Albania Bosnia and Herzegovina Kosovo Latvian Montenegro Serbia Ukraine Brazil Total number of samples

2 65 4 2 4 2 6 9 5 80 2 9 3 8 2 0 7 1 6 2 18 6 8 311 5 1 6 2 8 5 5 2 3 1 600

0.3 10.8 0.7 0.3 0.7 0.3 1.0 1.5 0.8 13.3 0.3 1.5 0.5 1.3 0.3 0.0 1.2 0.2 1.0 0.3 3.0 1.0 1.3 51.8 0.8 0.2 1.0 0.3 1.3 0.8 0.8 0.3 0.5 0.2 100

Meat and meat products 34 2 1 1 3 7 2 74

Fish and fish products

Milk and milk products

Bush meat

2 29 1

1

Egg

Honey 2

1 3 2 3 2 3 1

5 1

1 3

6 3

8 2 1 1 6 2 15 6 8 63 3 1 4 2 5 4 2 1 2 1 262

6

2

1 1

244 1

1

1

1

3

3 1 3 1 1 5

315

6

7

5

Africa. Asia. Europe. South America.


f

e

d

EU-VO 2073/2005, positive in 25 g; Campylobacter spp. was not present in any of the 600 samples. EU-VO 2073/2005: boiled sausages ≥100 cfu/g; minced meat ≥103 cfu/g; convenience food ≥104 cfu/g; raw meat (beef, pork, poultry) ≥5 × 104 cfu/g; raw sausages ≥104 cfu/g; dairy products ≥103 cfu/g. EU-VO 2073/2005: convenience food, boiled sausages, raw sausages ≥100 cfu/g; raw meat (beef, pork, poultry) ≥103 cfu/g; minced meat with spices ≥104 cfu/g; dairy products ≥103 cfu/g. Not yet reglemented, positive in 25 g. Referring to 262 meat and meat products. Total number of samples and percentage not meeting one or more criteria. c

b

15 (2.5) 7 (1.2) 17 (2.8) 66 (11) 8 (1.3) 91 (15.2) L. monocytogenesa Salmonella spp.a SA ≥b E. coli ≥c VTECd Total (%)f

a

3 (8.3) 2 (5.6) 1 (2.8) 11 (30.6) 0 (0.0) 14 (38.9) 8 (3.8) 3 (1.4) 3 (1.4) 13 (6.0) 3 (1.4) 25 (11.5) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (16.7) 0 (0.0) 1 (16.7) 0 (0.0) 1 (33.3) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (20) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (20) 3 (1.0) 1 (0.3) 13 (4.1) 42 (13.3) 5 (1.6) 50 (20.3) 11 (4.2) 5 (1.9) 4 (1.5) 24 (9) 3 (1.1) 39 (17.9) 2 (1.6) 0 (0.0) 2 (1.6) 7 (5.5) 0 (0.0) 9 (7.0) 0 (0.0) 4 (4.9) 5 (6.2) 18 (22.2) 0 (0.0) 23 (27.1) 2 (2.5) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 (2.5)

Bush Meat (6) Egg (7) Fish + fish products (5) Milk + milk prod. (315) Meat + meat Other n = 128 prod. (262) (%) Turkey n = 311 (%)

China n = 80 (%)

Africa n = 85 (%)

Food type Country of origin

No. of samples not meeting the criteria (%)

In total 262 meat and meat products with a total weight of 452.9 kg were seized. 28.2%, 24% and 13% of the samples were imported from China, Turkey and Egypt, respectively. 82.8% of all meat products were ready-to-eat, whereas 17.2% required further heat treatment or cooking (such as raw sausages, raw meat and fat). 37.3% and 25.8% of the readyto-eat meat products were heat-treated or dried. 35.8% and 12.9% of all meat samples could be identified as sausages and bacon, respectively. The respective animal species could be determined in 57.6% of all cases. 63, 50 and 37 samples were identified as poultry, beef and pork, respectively. Nine samples were mixed meats and only two samples comprised mutton or lamb. In contrast, 101 meat product samples could not be identified due to missing or incomplete labelling. The faecal indicator E. coli was identified in 24, coagulase-positive staphylococci (SA) in 4 meat and meat product samples, respectively, thereby exceeding maximum permissible values (Table 2). The determined ranges of E. coli and SA ranged from 1.3 × 102 cfu/g to 3.9 × 108 cfu/g and from 9 × 102 cfu/g to 1.5 × 105 cfu/g, respectively. Raw meat and raw animal fat samples had mean counts of 1.9 × 107 E. coli cfu/g. The highest concentration of E. coli, at 3.9 × 108 cfu/g was found in a raw sheep fat sample that originated from Egypt.

Criteria

3.6. Meat and meat products

Table 2 Samples not meeting the food-safety criteriaa and process hygiene criteriab,c of EU regulations and verotoxin-producing E. colid out of a total of 600 POAO.

Milk and milk products comprised 775.6 kg of the total samples. The main country of import was Turkey with 244 samples, followed by Egypt with 29 samples (Table 1). Of 315 milk products, 83.2% were cheese, 11.4% butter or ghee and 5.4% were fermented or processed milk products such as yoghurt, cream and sour cream. There were no samples of drinking milk (raw or heat-treated). The 262 cheese samples could be subdivided as follows: 47.3% was surface-ripened (soft, semi-hard and hard; 124/262), 37.7% were fresh cheeses (99/262), 7.6% brine cheeses (e.g. Feta; 20/262) and 7.3% stretched curd pasta filata-type cheeses, such as Mozzarella (19/262). Only 9.8% of milk products (31/315) were labelled and from labels it was possible to establish that these products were made from heat-treated (31) milk. 95.9% (302) and 86.7% (273) of all milk products met the European standard for SA and E. coli. All other samples showed a mean and maximum value of 1.9 × 10 5 cfu/g to 1.8 × 10 7 E. coli cfu/g and 1.0 × 106 to 2.5 × 108 SA cfu/g, respectively. Foodborne pathogens could be detected in 2.9% of the milk products (9/315); these included Salmonella spp. (n = 1), L. monocytogenes (n = 3) and VTEC (n = 5) (Table 2). One surface-ripened and two pasta filata-type cheeses tested positive for L. monocytogenes. Five fresh cheese products from Turkey contained verotoxin-producing E. coli (lor O2:H27; cream cheese O8:HNM; both stx1 positive; lor O178:H7; white cheese O8:HNM; white cheese O6:H10, all stx1 + stx2 positive). Finally, one sample from Istanbul, declared as “mozzarella in brine from granny”, contained not only Salmonella spp., but also 4 × 106 cfu/g of SA, and 2.4 × 102 cfu/g of E. coli (Table 3).

Honey (6)

3.5. Milk and milk products

11 (3.5) 3 (0.1) 10 (3.2) 41 (13.8) 8 (2.6) 57 (18.3)

Of the 600 samples of POAO, 52.5% (315) and 43.7% (262) involved milk and meat products, respectively. 0.8% (5), 1.2% (7) and 0.8% (5) of samples involved fish and sea-food, eggs and honey respectively. Only 1% (6) of samples could be categorized as bush meat (Table 1).

Ready-to-eat (217)

Type of processinge

3.4. Confiscated POAO — sample categories

Raw, for further processing (36)

Tinned food (9)

all samples were transported in sealed or unsealed plastic bags, respectively. 11.5% of samples were carried in plastic boxes and a smaller percentage of foodstuffs were wrapped in aluminium foil (4.3%), styrofoam cups (4.3%), paper bags (3.8%) or filled into plastic bottles (3%), aluminium cans (1.7%), aluminium bags (1.3%), wrapped in cotton bags (1.2%) or transported in glass (0.3%) or porcelain containers (0.2%).

5

0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)



6


Foodborne pathogens could be detected in 7.3% of the meat or meat products (19/262). Three, five and 11 cases contained VTEC, Salmonella spp. and L. monocytogenes, respectively (Table 2). Two meat samples from Egypt, two samples from Turkey and one from Tunisia were Salmonella spp. positive. L. monocytogenes could be detected in seven meat samples from Turkey, two from China, one from Albania and one from Armenia. Verotoxigenic E. coli were found in three samples from Turkey (Table 3).

hundred-year eggs), one from India and one from Russia. One sample originated from quail, one from duck and five were hens' eggs. Except for one raw fish sample that originated from Turkey, which was positive for L. monocytogenes, all other samples were microbiologically unremarkable.

3.7. Bush meat

Most L. monocytogenes isolates (11/15; 73.3%) originated from Turkish POAO samples, followed by two L. monocytogenes isolates from Chinese (13.3%) and both one from Albanian and Armenian imported food, respectively (each 6.7%). MLST analysis revealed that seven and eight L. monocytogenes isolates could be assigned to genetic lineages I/III and II, respectively (Fig. 1.1 and 1.2). The most common allelic profile was ST9 (26.7% of isolates), followed by ST1, ST87 and ST121 (each 13.3% of isolates). L. monocytogenes ST1, ST2, ST87 (genetic lineages I, III) and ST9, ST18, ST37 and ST121 were isolated from Albanian, Armenian, Chinese and Turkish raw meat and meat products. L. monocytogenes isolated from cheese samples (origin Turkey) were solely represented among genetic lineage I/III (ST1, ST3, and ST5; Fig. 1.1). Two L. monocytogenes isolated from Chinese poultry products were both ST87. One fish isolate (origin Turkey) was found to be ST121.

Over the reporting period a total of six bush meat samples were obtained. The bush meat samples originated from Nigeria (n = 3), South Africa (n = 2) and Ethiopia (n = 1) (Table 4). There were no instances of raw meat. Of the six samples, one (head of beef) was cooked, two were smoked and three (biltong, game, one not defined) were dried. Total bacterial counts ranged from 2.5 × 104 to 8.3 × 108 cfu/g. One dried meat sample that originated from Ethiopia was positive for Salmonella spp. and showed a total bacterial count of 9.8 × 106 cfu/g. 3.8. Eggs, fish and honey Seven egg, five honey and five fish samples could be investigated. Of the seven egg products, five originated from China (all traditional

3.9. MLST typing of L. monocytogenes isolates

Table 3 POAO contaminated with Salmonella spp., L. monocytogenes and verotoxigenic E. coli. Country of origin

Category of food

Type of food

Total weight in kg

Ethiopia Turkey Egypt

Bush meat Fish Meat

1.5 3.0 10.0

Turkey Albania Egypt Turkey Turkey Turkey

Meat Meat Meat prod. Meat prod. Meat prod. Meat prod.

Dried pieces of meat Raw pieces of fish Raw meat and innards (liver) Raw carcass poultry Raw pieces of meat Raw breaded meat Sausage Sausage Sausage, beef

Turkey

Meat prod.

Turkey

Number of pieces

Kind of packaging

Packaging material

Salmonella spp.

L. monocytogenes Verotoxin producing E. coli toxin serotype

1 2 4

op op op

Plastic bag Plastic box Plastic bag

Positive – Positive

– Positive –

– – –

– – –

2.0 0.5 1.1 2.0 1.0 1.0

1 1 10 2 18 1

op op op op op op

– – Positive – – –

Positive Positive – Positive Positive –

– – – – – stx1, stx2

– – – – – n.d.

Raw meat sticks, beef

1.2

3

sp

–

Positive

–

–

Meat prod.

Sausage

2.0

1

op

Plastic bag Plastic bag Plastic bag Plastic bag Plastic bag Aluminium foil and plastic bag Styrofoam cup, plastic foil Plastic bag

–

–

Tunisia Turkey China China Turkey Turkey Armenia Turkey

Meat prod. Meat prod. Meat prod. Meat prod. Meat prod. Meat prod. Meat prod. Meat prod.

Sausage Sausage Gizzard, cooked Duck pieces, cooked Sausages Sausages Sausages Sausages

2.0 1.0 0.7 0.5 1.5 3.0 1.5 1.0

4 1 1 1 1 2 2 1

op op op op op op op op

Plastic bag Plastic bag Plastic bag Plastic bag Plastic bag Plastic foil Paper and plastic bag Plastic bag

Positive Positive – – Positive – – –

– – Positive Positive – Positive Positive –

stx1, stx2, E-hly – – – – – – – stx2

Turkey Turkey Turkey

Meat prod. Meat prod. Milk prod.

15.0 0.5 3.0

5 2 1

op sp op

Paper and plastic bag Sealed plastic box Plastic bag

– – Positive

Positive Positive –

– – –

Turkey

Milk prod.

Sausages Sausages, beef Mozzarella in brine from granny Lor, whey cheese

O39: H48 – – – – – – – Orough: H7 – – –

5.0

2

op

Plastic box

–

–

Turkey Turkey

Milk prod. Milk prod.

Filata cheese White cheese

5.0 1.0

2 2

op op

– –

Positive –

Turkey Turkey


Filata cheese Cream cheese

5.0 10.0

4 2

op op

Plastic bag Aluminium foil and plastic bag Plastic bag Plastic box

– –

Positive –

Turkey

Milk prod.

Lor, whey cheese

1.3

1

op

Plastic bag

–

–

Turkey Turkey


Cheese White cheese

1.0 1.1

2 1

op op

Plastic bag Plastic bag

– –

Positive –

stx1, stx2, E-hly – stx1, stx2, E-hly – stx1, eaeA, E-hly stx1, eaeA, E-hly – stx1, stx2, E-hly

O178: H7 – O6:H10 – O8: HNM O2:H27 – O8: HNM

sp original sales packaging like canned items or sealed plastic bags. op packaged by passenger, mostly wrapped in paper or plastic bags.


– – 1.80E+06 – b100 b10 Plastic foil op 2 Kudu London Cape Town

n.d. not defined. op packaged by passenger, mostly wrapped in paper or plastic bags.

0.7

op op 1 3 1.5 0.3 n.d. n.d. Addis Abeba – Cape Town London

Ethiopia South Africa South Africa

Amsterdam

Beef Lagos Nigeria

Amsterdam

n.d. Lagos Nigeria

Dried pieces of meat Biltong, cured, dried mixed meet Sliced, dried game meat

op 2 1.5

op 2 0.8

–

– – – – – – 9.80E+06 positive 8.30E+08 – 2.88E+03 b100 b10 b100

– – 4.90E+08 – 8.00E+01 b100

–

– – 5.90E+04 – b100 b10

–

– – 2.51E+04 – b100 b10

Newspaper and plastic bag Newspaper and plastic bag Newspaper and plastic bag Plastic bag Paper bag op 2 0.7

Dried and smoked pieces of meat Dried and smoked pieces of meat Head meat, cooked Amsterdam

n.d. Lagos Nigeria

Non-stop/ stop over Departure airport

Animal species

Type of food

Total weight in kg

Number of pieces

Kind of packaging

Packaging material

TBC cfu/g SA cfu/g E. coli cfu/g

Salmonella spp.

7

Country of origin

Table 4 Confiscated bush meat samples.

Increased importation of foods to the EU in general, and exotic produce in particular, albeit legitimately through regular import channels, carries with it the risk of zoonotic transmission and potential disease outbreak. In the USA, for instance, the Centers for Disease Control and Prevention observed that over the period 2005–2010 there was a positive correlation between increasing volumes of imported foods and the number of disease outbreaks (Gould et al., 2012). As yet no reliable assessments can be made for the EU regarding the significance that illegally imported animals or POAO contribute to the total number of known disease outbreaks. Instead, what is available is a limited number of well-documented cases that repeatedly illustrate the relevance of the problem. A well-documented case-controlled study by Kapperud et al. (1998) concerned a Salmonella typhimurium outbreak in humans, which incriminated illegal importation of poultry meat by travellers. Two similar cases of trichinellosis outbreaks were reported from Austria and Bavaria, where illegally imported meat products by immigrant workers from Bosnia-Herzegovina and Rumania (at that time non-EU countries), respectively, were confirmed as causal factors (Lechner et al., 2012; Nöckler et al., 2007). In both cases traveller ignorance of the risks associated with illegal food imports, combined with failed border control checks, resulted in disease outbreak. In the latter incident, the cured paprika sausage confiscated at the patients' household had a larval density (as assessed by in vitro digestion) as high as 441 larvae/g (Nöckler et al., 2007). For the EU, outbreak investigation data related to cases attributable to illegally imported animals or POAO are scarce, fragmented and not readily retrievable. With special permission of the Austrian health authorities, the luggage of 61,355 passengers from customs applicable flights arriving at VIE airport was inspected. Over a period of eight months 1473 POAO samples were confiscated. A total of 600 samples were suitable for microbiological analysis. Foodborne pathogens could be detected in 5% (30/600) of all samples. The present study revealed that the majority of the 600 samples investigated originated by far from Asia (80.5%), Turkey (51.8%) followed by China (13.3%). POAO from these countries were mostly represented by milk products, followed by meat and meat products. Africa was the second main source of seized POAO samples (14.2%), which were mostly meat and meat products (45.2%), closely followed by milk products (42.4%). Interestingly, the foodborne pathogen VTEC had highest prevalence and was exclusively found in seizures from Turkey (2.6%; 8/311). These data correspond well with the results from Cadirci et al. (2006) who found 2.5% verotoxin producing E. coli O157 in 200 beef and meat ball samples produced in Turkey. In the current study, the highest prevalence of L. monocytogenes was in fish (20%), in raw foods that required further processing (8.3% of samples) and in meat and meat products (4.2% of samples). According to the national report on zoonoses and zoonotic agents in Austria the prevalence of L. monocytogenes in milk, milk products or cheeses was 0.2%, in fish and fish products 4.6% and in meat and meat products 5.4% (AGES, 2012). L. monocytogenes STs isolated in the current study, mainly isolated from Turkish and Chinese POAO, corresponded to the worldwide most prevalent STs and clonal complexes 1, 2, 3, 5, 9, and 121 (ChenalFrancisque et al., 2011). Generally, ST9 was the most prevalent allelic profile, which was mainly isolated from Turkish meat samples. A high ST9 prevalence was also found in Spanish, Italian and Chinese meat plants and meat products (Martín et al., 2014; Parisi et al., 2010; Wang et al., 2012). Recently, L. monocytogenes ST87 isolates resulted in a foodborne outbreak in Spain in 2013 (Institute Pasteur Listeria monocytogenes MLST database), these were predominant among

L. monocytogenes VTEC Campylobacter spp.

4. Discussion

–



8


Fig. 1.1 and 1.2. Multi-locus sequence typing of 15 L. monocytogenes isolates from food confiscated from air passenger luggage. The sequence types were clustered according to the abcz housekeeping gene using a minimum spanning tree (MST) tool available from the Institute Pasteur MLST database (http://www.pasteur.fr/recherche/genopole/PF8/mlst/). Numbers within circles denote the corresponding ST within genetic lineage I, III (Fig. 1.1) and II strains (Fig. 1.2). Numbers surrounded by red dotted lines represent the ST found in the current study. L. monocytogenes sample origins are included in each MST marked by black dotted lines. Coloured zones surrounding groups of STs represent clonal complexes (CC) differing by only one gene from another member of the group.

Chinese food isolates (Wang et al., 2012), and were also isolated in this study from two Chinese poultry products. Prevalence of Salmonella spp. was highest in bush meat (16.7% of samples; 1/6), followed by other confiscations from Africa (4.9% of samples; 4/81). African products also showed the highest prevalence

of E. coli (22.2%; 18/81), followed by bush meat (16.7%; 1/6) and milk and milk products in general (13.3%; 42/315). The findings of the presented study illustrate the potential public health risks associated with illegal imports of food items, many of which were poorly packaged, unlabelled and unrefrigerated. Additionally,



and most relevantly, these imports to the EU are potential harbingers of novel strains of known pathogens. Similar observations concerning the illegal import of traditional Mexican cheeses have been reported from the USA (Sandoval et al., 2012). These authors suggest that longstanding cultural practices and a lack of knowledge about what is and what is not legal make it difficult for travellers to acknowledge the associated risks. This is a plausible foundation for our observational experience in Europe. The 1957 Treaty of Rome and its revisions (1986, 1992 and 1997) focus on the internal EU market without borders, where products originating from a third country and entering the EU must be ‘free to move’ within member states. This is a principle that operates in tandem with the goal of biosecurity, which aims to reduce the amount of illegal food or animals imported by travellers arriving from third countries, or sent by post. Customs posts at international borders and airports are crucial to implementing these controls. This has resulted in a series of actions organised in airports, ports and road-entry points in cooperation with the Directorate General for Taxation and Customs Union of the European Commission and the veterinary and customs authorities in member states. The aim is to inform the public about the rules governing personal consignments of POAO and pets (Noordhuizen et al., 2013). However, these biosecurity measures are only superficially effective, according to Noordhuizen et al. (2013). Neither are they sufficient for properly protecting European food animal production sectors nor for safe-guarding public health. Illegal importation of animals or POAO still occurs and sometimes to a large extent. NAO (2005) reported that an estimated 12,000 tons of meat and meat product were illegally imported to the UK. This is particularly relevant to the EU as animal and/or human disease outbreaks occurring within its borders represent a serious threat. Although the precise sources of these outbreaks are usually unknown, it is often assumed they could indeed be related to deficient biosecurity measures at border controls (Noordhuizen et al., 2013). Bush meat is a unique challenge to EU biosecurity. It is raw, smoked or partially processed meat that originates from a variety of animals, including non-human primates, bats, antelopes and rodents, typically obtained from forest areas where there is an abundance of wildlife. Many of these species are listed on the Washington Convention on International Trade in Endangered Species of Wild Fauna and Flora. Bush meat is not only imported for personal consumption, it is also part of a lucrative organised trade in multiple exotic species, demanding high prices that associate the meat with luxury status. In the present study bush meat was associated with Salmonella spp. contamination (Table 4). Six samples (7.1%) of 85 from Africa could be identified as bush meat, representing 1% of all seized samples (6/600). Most bush meat samples originated from Nigeria (three samples), followed by South Africa (two) and Ethiopia (one). Bush meat was commonly encountered as dried meat that was awkwardly wrapped in newspaper or unsealed plastic bags. In comparison, another study of bush meat importation into Europe estimated in 2010 that 5 tons were imported weekly at the Charles de Gaulle Airport, Paris, France (Chaber et al., 2010). This very high amount may be a consequence of former French colonial connections and resulting immigration to France and ongoing return visits. Likewise, importation of bush meat into the US is prohibited. However, illegal importation persists as bush meat is traditionally consumed by US immigrants from implicated regions. Bair-Brake et al. (2014) report a US study where a total of 543 bush meat items were confiscated. Half of these items were identified as rodents and most frequently they originated from Africa. Clearly, unregulated trade in wildlife species, such as those appearing as bush meat, raises serious ethical and safety concerns. It is unrealistic to expect implementation of a fail-safe biosecurity system at all points of entry to the EU. Many confounding factors exist and to date there is only a limited number of case reports from which

9

relevant experience can be built. Currently available source attribution data suggest that increased global trade and travel activities will be major contributors to future outbreaks of animal and human disease (Vagsholm and Smulders, 2012), presumably regardless of measures taken. Unfortunately, a systematic, evidence-based risk assessment that would tolerate cost-effective risk management decisions will not become available until results of more targeted baseline studies are published. Merely relying on economic or other mathematical simulation models would seem far from prudent, as these are essentially non-specific tools. At the same time, travellers must understand and learn to accept why import prohibitions are in place. Acknowledgement A.S. was supported by the EU KBBE project “Protection of consumers by microbial risk mitigation through combating segregation of expertise”, Grant Agreement no. 265877. Thanks to the Austrian Agency for Health and Food Safety (AGES) for excellent cooperation and providing the Austrian L3 High Security Laboratories for investigations. Furthermore Dr. Cameron McCulloch is thanked for assistance with the manuscript. References Austrian Agency for Health and Food Safety (AGES), 2012. Report on zoonoses and zoonotic agents in Austria 2012. Available from http://www.ages.at/uploads/media/ Report_on_Zoonoses_and_zoonotic_Agents_in_Austria__2012.pdf (accessed on 29 April 2014). Bair-Brake, H., Bell, T., Higgins, A., Bailey, N., Duda, M., Shapiro, S., Eves, H.E., Marano, N., Galland, G., 2014. Is that a rodent in your luggage? A mixed method approach to describe bush meat importation into the United States. Zoonoses Public Health 61, 97–104. Border, P.M., Howard, J.J., Plastow, G.S., Siggens, K.W., 1990. Detection of Listeria species and Listeria monocytogenes using polymerase chain reaction. Lett. Appl. Microbiol. 11, 158–162. Bubert, A., Hein, I., Rauch, M., Lehner, A., Yoon, B., Goebel, B., Wagner, M., 1999. Detection and differentiation of Listeria spp. by a single reaction based on multiplex PCR. Appl. Environ. Microbiol. 65, 4688–4692. Cadirci, O., Siriken, B., Inat, G., Kevenk, T.O., 2006. The prevalence of Escherichia coli O157 and O157:H7 in ground beef and raw meatball by immunomagnetic separation and the detection of virulence genes using multiplex PCR. Int. J. Food Microbiol. 106, 338–342. Chaber, A.L., Allebone-Webb, S., Lignereux, Y., Cunningham, A.A., Rowcliffe, J.M., 2010. The scale of illegal meat importation from Africa to Europe via Paris. Conserv. Lett. 3, 317–321. Chenal-Francisque, V., Lopez, J., Cantinelli, T., Caro, V., Tran, C., Leclercq, A., Lecuit, M., Brisse, S., 2011. Worldwide distribution of major clones of Listeria monocytogenes. Emerg. Infect. Dis. 17, 1110–1112. EuroKnow, 2012. www.euro-know.org/europages/dictionary/f.htm (Available from: accessed on 4 April 2014). European Commission, 1991. Council Directive 91/496/EEC of 24 September 1991 laying down the principles governing the organization of veterinary checks on animals entering the Community from third countries. Off. J. Eur. Union L (268/56-67). European Commission, 1997. Council Directive 97/78/EC of 18 December 1997 laying down the principles governing the organization of veterinary checks on products entering the Community from third countries. Off. J. Eur. Union L24, 9–30. European Commission, 2004. Commission Regulation (EC) No 136/2004 of 22 January 2004 laying down procedures for veterinary checks at Community border inspection posts on products imported from third countries. Off. J. Eur. Union L21, 11–23. European Commission, 2007. Commission Decision 2007/275/EC from 4 May 2007 concerning lists of animals and products to be subject to controls at border inspection posts under Council Directives 91/496/EEC and 97/78/EC. Off. J. Eur. Union L (116/9-33). European Commission, 2009a. Commission Regulation (EC) No 206/2009 of 5 March 2009 on the introduction into the Community of personal consignments of products of animal origin and amending Regulation (EC) No 136/2004. Off. J. Eur. Union L77/1. European Commission, 2009b. Commission Regulation (EC) No 1069/2009 of 21 October 2009 laying down health rules as regards animal by-products and derived products not intended for human consumption. Off. J. Eur. Union L300, 1–33. European Commission, 2012. International Affairs — Import Conditions. Available from: http://ec.europa.eu/food/international/trade/index_en.htm. Food and Veterinary Office (FVO), 2013. http://ec.europa.eu/food/fvo/index_en.cfm (Available from: accessed on 4 April 2014). Gould, L., Morse, D., Tauxe, R.V., 2012. Foodborne disease outbreaks associated with food imported into the United States, 2005–2010. International Conference on Emerging Infectious Diseases, 2012: Posters and Oral Presentation Abstract. Emerging Infectious Diseases, pp. 295–296 (March, Available from: http://www.cdc.gov/EID/ pdfs/ICEID2012.pdf; accessed on 4 April 2014).


10


Institute Pasteur Listeria monocytogenes MLST database, e. http://www.pasteur.fr/ recherche/genopole/PF8/mlst/Lmono.html (accessed: 25.01.2014). ISO, 1999a. ISO 6888-1:1999 Microbiology of Food and Animal Feeding Stuffs — Horizontal Method for the Enumeration of Coagulase-positive Staphylococci (Staphylococcus aureus and Other Species), Part 1: Technique Using Baird–Parker Agar Medium. International Organization for Standardization, Geneva, Switzerland. ISO, 1999b. 6888-2:1999 Microbiology of Food and Animal Feeding Stuffs — Horizontal Method for the Enumeration of Coagulase-positive Staphylococci (Staphylococcus aureus and Other Species), Part 2: Technique Using Rabbit Plasma Fibrinogen Agar Medium. International Organization for Standardization, Geneva, Switzerland. ISO, 2001. 16649-2:2001 Microbiology of Food and Animal Feeding Stuffs — Horizontal Method for the Enumeration of Beta-glucuronidase-positive Escherichia coli, Part 2: Colony-count Technique at 44 Degrees C Using 5-bromo-4-chloro-3-indolyl beta-Dglucuronide. International Organization for Standardization, Geneva, Switzerland. ISO, 2002. ISO 6579:2002 Microbiology of Food and Animal Feeding Stuffs — Horizontal Method for the Detection of Salmonella spp. International Organization for Standardization, Geneva, Switzerland. ISO, 2003. ISO 4833:2003 Microbiology of Food and Animal Feeding Stuffs — Horizontal Method for the Enumeration of Microorganisms — Colony-count Technique at 30 Degrees C. International Organization for Standardization, Geneva, Switzerland. ISO, 2004. 11290-1:2004 Microbiology of Food and Animal Feeding Stuffs — Horizontal Method for the Detection and Enumeration of Listeria monocytogenes, Part 1: Detection Method. International Organization for Standardization, Geneva, Switzerland. ISO, 2006. ISO 10272: 2006 Microbiology of Food and Animal Feeding Stuffs — Horizontal Method for Detection and Enumeration of Campylobacter spp. International Organization for Standardization, Geneva, Switzerland. ISO, 2012. ISO/TS 13136:2012 microbiology of food and animal feeding stuffs — real-time polymerase chain reaction (PCR)-based method for the detection of food-borne pathogens. Horizontal Method for the Detection of Shiga Toxin-producing Escherichia coli (STEC) and the Determination of O157, O111, O26, O103 and O145 SerogroupsInternational Organization for Standardization, Geneva, Switzerland. Kapperud, G., Lassen, J., Hassevedt, V., 1998. Salmonella infections in Norway: descriptive epidemiology and a case–control study. Epidemiol. Infect. 121, 569–577. Karner, R., 2012. Personal communication. Lechner, A., Kraus, J., Hoppe, U.C., Glawischnig, W., Auer, H., Allerberger, F., 2012. Outbreak of human trichinellosis, Austria 2010. Wien. Tierarztl. Monatsschr. Suppl. 2, 20–23. Martín, B., Perich, A., Gómez, D., Yangüela, J., Rodríguez, A., Garriga, M., Aymerich, T., 2014. Diversity and distribution of Listeria monocytogenes in meat processing plants. Food Microbiol. 44, 119–127. NAO, 2005. National Audit Office. Press Notice 27-05 of 23 March 2005, Available from: www.nao.gov.uk/, accessed: 4 April 2014. Nöckler, K., Wichmann-Schauer, H., Hiller, P., Müller, A., Bogner, K., 2007. Trichinellosis outbreak in Bavaria caused by cured sausage from Romania, January 2007. Euro

Surveill. 12, 3254 (Available http://www.eurosurveillance.org/ViewArticle.aspx? ArticleId=3254; accessed on 4 April 2014). Noordhuizen, J., Surborg, H., Smulders, F.J.M., 2013. On the efficacy of current biosecurity measures at EU borders to prevent the transfer of zoonotic and livestock diseases by travellers. Vet. Q. 33, 161–171. Parisi, A., Latorre, L., Normanno, G., Miccolupo, A., Fraccalvieri, R., Lorusso, V., Santagada, G., 2010. Amplified fragment length polymorphism and multi-locus sequence typing for high-resolution genotyping of Listeria monocytogenes from foods and the environment. Food Microbiol. 27, 101–108. Protection of consumers by microbial risk mitigation through combating segregation of expertise 2012. Available from: http://promisenet.wordpress.com (accessed on 4 April 2014). Ragon, M., Wirth, T., Hollandt, F., Lavenir, R., Lecuit, M., Le Monnier, A., Brisse, S., 2008. A new perspective on Listeria monocytogenes evolution. PLoS Pathog. 4, e1000146.#. Rahn, K., De Grandis, S.A., Clarke, R.C., McEwen, S.A., Galán, J.E., Ginocchio, C., Curtiss, R., Gyles, C.L., 1992. Amplification of an invA gene sequenceof Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Mol. Cell. Probes 6, 271–279. Reischl, U., Youssef, M.T., Kilwinski, J., Lehn, N., Zhang, W.Z., Karch, H., Strockbine, N.A., 2002. Real-time fluorescence PCR assays for detection and characterization of Shiga toxin, intimin, and enterohemolysin genes from Shiga toxin-producing Escherichia coli. J. Clin. Microbiol. 40, 2555–2565. Sandoval, M., Corona-Luevanos, A., Puentes, F., Escobedo, L., Escobedo, M., 2012. A bacteriological assessment of imported cheese from Mexico-El Paso. International Conference on Emerging Diseases, 2012: Poster and Oral Presentations. Emerging Infectious Disease, pp. 215–216 (March, Available from: http://www.cdc.gov/EID/ pdfs/ICEID2012.pdf; accessed on 4 April 2014). Vagsholm, I., Smulders, F.J.M., 2012. Symptomless carriers and the rationale for targeted risk management strategies. Wien. Tierarztl. Monatsschr. 99, 272–277. Walsh, P.S., Metzger, D.A., Higuchi, R., 1991. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques 10, 506–513. Wang, G., Clark, C.G., Taylor, T.M., Pucknell, C., Barton, C., Price, L., Woodward, D.L., Rodgers, F.G., 2002. Colony multiplex PCR assay for identification and differentiation of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus subsp. fetus. J. Clin. Microbiol. 40, 4744–4747. Wang, Y., Zhao, A., Zhu, R., Lan, R., Jin, D., Cui, Z., Wang, Y., Li, Z., Wang, Y., Xu, J., Ye, C., 2012. Genetic diversity and molecular typing of Listeria monocytogenes in China. BMC Microbiol. 12, 119. WHO, R., 2010. Guidance on regulationsfor the Transport of Infectious Substances 2011–2012. Available at: https://www.biosafety.moh.gov.sg/home/uploadedFiles/ Common/WHO_Guidance_on_regulations_for_transport_of_Infectious_Substances. pdf; accessed: 13. August 2014.


Prevalence of Foodborne Pathogens in Freshwater Fish in Latvia.

Bacterial Quality and Prevalence of Foodborne Pathogens in Edible Offal from Slaughterhouses in Korea.

Characterization of illegal food items and identification of foodborne pathogens brought into the European Union via two major German airports.

Stress adaptation in foodborne pathogens.

Bacteriophage biocontrol of foodborne pathogens.

Essential oils from herbs against foodborne pathogens in chicken sausage.

Microbial assessment and prevalence of foodborne pathogens in natural cheeses in Japan.

Antibiotic susceptibility and prevalence of foodborne pathogens in poultry meat in Romania.

Detection of foodborne bacterial pathogens from individual filth flies.

Prevalence of Foodborne Pathogens in Cooked Meat and Seafood from 2010 to 2013 in Shandong Province, China.

Microbiology and foodborne pathogens in honey.

Co-occurrence of free-living protozoa and foodborne pathogens on dishcloths: implications for food safety.

Effect of atmospheric pressure plasma jet on the foodborne pathogens attached to commercial food containers.

Molecular Detection of Foodborne Pathogens: A Rapid and Accurate Answer to Food Safety.

Comparative Resistance of Bacterial Foodborne Pathogens to Non-thermal Technologies for Food Preservation.

Detection of Foodborne Pathogens and Mycotoxins in Eggs and Chicken Feeds from Farms to Retail Markets.

Application of bacteriophages for detection of foodborne pathogens.

Surface adhesins and exopolymers of selected foodborne pathogens.

Epidemiology, Detection, and Control of Foodborne Microbial Pathogens.

A foodborne outbreak of gastroenteritis involving two different pathogens.

Immunological methods for detection of foodborne pathogens and their toxins.

Foodborne bacterial pathogens recovered from contaminated shawarma meat in northern Jordan.

Multiplexed detection of foodborne pathogens based on magnetic particles.

Antibacterial effect of silver nanoparticles against four foodborne pathogens.