Slov Vet Res 2016; 54 (4): 195-204 UDC 614.31:637.56:578 Original Research Article VIRAL CONTAMINATION IN MUSSEL PRODUCTION CHAIN ON THE SLOVENIAN COASTLINE Mateja Ambrožič1, Urška Henigman2, Mojca Jevšnik3, Andrej Kirbiš2, Peter Raspor1 1Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, institute for Food Hygiene, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, 3Department of Sanitary Engineering, Faculty of Health Sciences, University of Ljubljana, Zdravstvena pot 5, 1000 Ljubljana, Slovenia Corresponding author, E-mail: peter.raspor@guest.arnes.si Summary: Noroviruses (NoVs) and Hepatitis A virus (HAV) are significant agents of foodborne human viral illness worldwide, both as agents of greatest concern from the consumption of shellfish. In the framework of official national monitoring shellfish samples have been collected since 2013 to determine the spread of NoVs and HAV. Contamination of shellfish samples with NoVs varied from 25% in year 2014 to 40% in year 2015. HAV was not found in any of the analyzed samples, which correlates with the low number of human infections in Slovenia. Alongside official surveillance, semi-structured interviews were carried out with local shellfish farmers regarding this subject. The qualitative analysis highlighted that Slovenian shellfish farmers are aware of food safety hazards, especially associated with hazards to primary production, but only in the context of Escherichia colias an indicator microorganism, and not in the concept of viral food safety. Despite detected foodborne viruses in shellfish on the Slovenian market, local farmers are not aware of or do not recognize foodborne viruses as distinctive food safety hazards. It can be concluded that local farmers possess knowledge and information of critical points in the mussels food supply chain connected to food safety in general. However, in particular, they are not familiar with viruses that represent barrier and consequently critical point to food safety management practices. Training and education on all important aspects of viral food safety according to the current recommendations is strongly recommended for all actors within the shellfish supply chain. Key words: mussel farmer; Slovenia; official control; food safety; foodborne viruses; semi-structured personal interviews Introduction Mariculture is a traditional activity in the Primorska region. Mediterranean mussels (Mytilus galloprovincialis) are the main harvested species and, of a smaller quantity, also warty venus (Venus verrucosa). Mussel farming takes place in a standard manner in lines of floating buoys linked together, with longline nets hung from them. In Slovenia, within 46.6 km of coastline there are three official harvesting areas of Mediterranean Received: 10 November 2015 Accepted for publication: 21 April 2016 mussels: Seča, Strunjan, and Debeli rtič, with 56 registered shellfish farmers on a total area of 46 ha. In 2013, 231 persons were involved in aquaculture activities in Slovenia, and only 39 persons were involved in marine fish and shellfish farming. The aquaculture sector in Slovenia is mainly characterized by small self-employed family farms, most of which have one employee, and some are assisted by unpaid family members. Total shellfish production in 2013 was 311 tonnes, and current production covers mainly the needs of the domestic market. The major cultured shellfish species, Mediterranean mussel, accounts 196 M. Ambrozic, U. Henigman, M. Jevsnik, A. Kirbis, P. Raspor for 83 percent of total mariculture production in Slovenia (1, 2). Next to that there were about 153 tons of imports of mussels in Slovenia but only 23 tons of export, what is a negligible quantity in alimentation compared to consumption of fresh fruit and vegetables, which are also recognized as vulnerable to viral contamination. Yet, the level of shellfish consumption by country is very different. In some countries, the mussel consumption per capita is over 3 kg per year, while it is not even part of the local diet in others (3). Per capita consumption of fresh fruit in Slovenia was in 2013 75 kg and 73 kg of fresh vegetables. However, there were no contaminated samples with NoVs or HAV of fresh produce found within Slovenian national monitoring (27). Shellfish specialties are commonly part of the culinary and gastronomic specialties only along the Slovene coast and are generally prepared and served in restaurants. The filter-feeding nature of bivalves and the traditional way of consuming them (often raw or slightly cooked) make shellfish one of the most common vehicles of viral foodborne illness. Shellfish are filter-feeding animals, which ingest and accumulate human pathogens (5). Lees (6) reported that shellfish grown in sewage-polluted waters tend to bio-accumulate environmentally stable enteric viruses. Processing interventions such as depuration do not eliminate viral particles (7), and food consumption practices increase the health risk related to shellfish consumption. The increasing amount of data on virus detection in shellfish (8, 9, 10) and shellfish-borne viral outbreaks (11) indicates the necessity of a constant surveillance system in European countries, including Slovenia (12). The management of the harvesting areas continues to rely uniquely on bacterial standards, such as Escherichia coli, despite the proven fact of being unreliable tools to indicate the viral presence in harvesting areas or to control the efficiency of the process, such as depuration (13, 14). From a virological point of view, shellfish safety continues to be a sanitation challenge and to protect consumers the EU strives towards establishing legislation on this matter. With better diagnostic technology and investigative epidemiology, it is now accepted that enteric viruses are major contributors to foodborne disease as well. Enteric viruses are transmitted through contaminated food, but also in combination with person-to-person contact or through environmental contamination. They have been increasingly recognized a significant cause of foodborne disease, despite the measures already in place, mainly targeted at reducing bacterial contamination, because of the increasing consumption of ready-to-eat foods, raw and/ or minimally processed shellfish, fruits, and vegetables. This is because products are often imported from areas lacking strict hygienic measures, they are often eaten uncooked, and they often come into contact with potentially contaminated animal manure, water, ice, human hands and surfaces from the "farm-to-table" continuum (10). Most foodborne viruses are more resistant (15) than bacteria to commonly used control measures, (e.g. refrigeration, freezing, pH, drying, UV radiation, heat, pressure, disinfection, etc.). There are currently no effective, realistic and validated risk management options to eliminate viral contamination prior to consumption without changing the normally desired characteristics ofthe food. Because of concerns about virus persistence during food processing, effective control strategies need to focus on the prevention of contamination. From the limited available information, foodborne viruses have a low infectious dose and are dispersed in stool or emesis in high numbers. Only a few viral/infectious particles are needed to cause an infection that may lead to illness (10, 15, 17). Shellfish aquaculture is a marine-based industry that is affected by other land users such as tourism, recreation, forestry, agriculture, and urban development. In many cases, the public is unaware of the detrimental impact their activities have on the aquaculture sector and, consequently, also on shellfish food safety. Food safety embraces the absence or acceptable and safe levels of contaminants, adulterants, naturally occurring toxins or any other substances that can make food dangerous to human health. Microbial food safety is considered a significant public health issue but historically has focused mostly on the control of bacterial contamination; however, enteric viruses have been increasingly recognized as an important cause of foodborne disease, and control measures are being developed (16, 17). The food supply chain from stable to table includes activities such as production, processing, distribution, retail, packaging and labeling of foodstuffs, which are governed by a mass of laws, regulations, codes of practice and guidance. Nowadays, the distance that food travels from producer to consumer has increased as a result of globalization in the food Viral contamination in mussel production chain on the Slovenian coastline 197 trade. Moving these food products safely and efficiently from farm to fork requires a highly coordinated series of links in a long chain of trading partners. Food miles, as a term that refers to the distance food is transported from where it is grown or raised to where it is purchased by a consumer, is part of the broader issue of sustainability that deals with a large range of environmental, social and economic issues. Therefore, keeping safety and quality along the food supply chain has become a significant challenge, whereas good traceability systems, defined as the ability to trace and follow a food, feed, food-producing animal or substance intended to be, or expected to be incorporated into a food or feed, through all stages of production, processing and distribution (18), help to minimize the production and distribution of unsafe or poor quality products. Epidemiology of foodborne viruses Although shellfish consumption can contribute to a healthy diet they are often associated with outbreaks of foodborne disease. Viral foodborne outbreaks associated with shellfish consumption have occurred in many countries (11) despite existing strategies to prevent contamination. They are often attributed to water contamination by sewage and/or during processing and serving. According to epidemiological evidence, NoVs as the predominant agents of nonbacterial gastroenteritis in humans along with Hepatitis A virus (HAV), both as agents of greatest concern from the consumption of shellfish, are important agents of foodborne human viral illness worldwide (17, 19, 20, 21, 22, 23). Of the approximately 600 million cases of illness caused by foodborne hazards in 2010 worldwide, infectious agents that cause diarrheal diseases accounted for the vast majority (550 million), in particular noroviruses (120 million cases) and Hepatitis A virus 14 million cases (24). A total of 5251 foodborne outbreaks were reported in 2014 in the EU (20) within the framework of member states' national monitoring. In 2014, food-borne viruses were, for the first time, identified as the most commonly detected causative agent in the reported food-borne outbreaks. 1070 food-borne outbreaks caused by viruses were reported in 2014, implicated 11740 cases, 2486 hospitalizations and 2 deaths. In strong-evidence outbreaks caused by viruses, 'crustaceans, shellfish, mollusks and products thereof was the most commonly implicated food vehicle (44.7% of outbreaks), followed by 'buffet meals' (15.8% of outbreaks), 'mixed food' (13.2%) and 'fruit' and 'berries and juices' (both 5.3%). The place of exposure most frequently reported was 'restaurant, café, pub, bar, hotel', followed by the household. Norovirus was the most commonly reported virus implicated in the strong-evidence outbreaks and accounted for 97.6% of cases. National statistics on foodborne viral disease are not easily available and, where present, likely to reflect significant under-reporting (17), because there is a lack of systematic surveillance for foodborne viral disease (25). Considering the scientific opinion from the EFSA, RASFF notifications and results of official controls, since 2013 the Slovenian National Zoonoses Monitoring Programme has included food sampling for the presence of NoVs and HAV in live shellfish at the retail level and distribution of local and foreign origin, which are recognized as potentially zoonotic viruses (26). In Slovenia, the Zoonoses Monitoring Programme (27) has been conducted at the national level since 1985. It is designed for the systematic collection, monitoring, analysis and communication of data on the emergence of zoonosis, zoonotic agents, and related antimicrobial resistance and comprises the recently emerging zoonotic agents, including foodborne viruses. The ultimate purpose is to capture high-quality information about infections in humans as well as in animals and the contamination of foods, providing important information that is integrated across sectors. It should provide a fundamental basis for making public health decisions with actions for reducing the risks to public health, document the impact of an intervention, track progress towards specified goals, and elucidate the epidemiology of health problems. The results obtained within Slovenian official national monitoring have shown that live shellfish, purchased at retail stores in Slovenia during independent sampling times throughout the year were contaminated with NoVs, but HAV was not found in any of the samples analyzed (Table 1), which correlates with the low number of human infections in Slovenia (Table 2). In studies, contamination of mussels' samples varies from 16.9% (9), to 34.4% NoVs in Italy (28), to 35.0% of contaminated mussels in France (29). Henigman et al. (8) reported 198 M. Ambrožič, U. Henigman, M. Jevšnik, A. Kirbiš, P. Raspor Table 1: Presence of NoVs and HAV in live shellfish samples within Slovenian official surveillance Year Virus NoVs HAV 2013 5/17 (29%) 0/15 2014* 3/12 (25%) 0/12 2015* 3/10 (40%) 0/10 ^Preliminary results Table 2: Reported infections caused by NoVs and HAV in humans in Slovenia from 2007-2014 (27) N° of cases Year 2007 2008 2009 2010 2011 2012 2013 2014* NoVs 1094 1043 1393 2012 2231 1611 2146 1316 HAV 15 17 12 9 12 11 23 10 ^Preliminary results practices are segregated along the food supply chain and are not connected to a comprehensive system, resulting in the existence of exposure to potential of food hazards, especially emerging hazards, such as viruses (12, 30). The development in different areas within the food production chain and in particularly in technological and technical means is moving very quickly. We encounter innovations in materials, and supporting measures almost daily. Consequently, the gap between knowledge and skills is widening. We are willing to accept the paradigm that drifting is the most dangerous challenge in analytical instruments. However, it is also extremely influential in technological practices. It occurs side by side with "industrial blindness", which develops as a personal characteristic of employees who do not see particular items although they are commonly present in routine operations. Consumers play an important role in the transmission of hazards, including viruses. Implemented viral food safety guidelines (12) are not purposely designed for informing consumers, although studies in recent years have highlighted gaps in food safety knowledge and some critical safety violations regarding food handling at home (31, 32, 33, 34, 35, 36). Consumer behaviour and attitudes toward food safety have shown that the levels of understanding, motivation and trust need to be further cultivated, and their training and informing due to changes in lifestyle and food that mussels collected in Slovenian coastal waters were contaminated with NoVs, the highest at Debeli Rtič (25.9%), 21.2% in Strunjan, and only 8.1% in the Seča harvesting area. The difference in positive results is interlinked to the location of harvesting areas due to sea current, dense shipping, and the influx of streams and rivers. Control and prevention of foodborne viruses Virus contamination as a consequence of human handling can occur at any stage of food production, processing, and even preparation. At present, we are faced with insufficient knowledge and awareness of food safety issues among food handlers and accompanied by consumers being insufficiently informed about food safety principles in the home. Today, we manage food safety through good practices at different levels within a food supply chain that can be described as a network of food-related businesses involved in the creation and consumption of food products that move from farm to table and are linked by information, material, and capital flows. Good practices are described in several different codes of practice designed by producers' organizations, importers and retailer's consortia and government bodies at different levels of production, processing and consumption within the food supply chain. All current active Viral contamination in mussel production chain on the Slovenian coastline 199 consumption patterns encouraged (30, 37, 38, 39). EFSA reported (20) that viral foodborne outbreaks most frequently occurred in 'restaurant, café, pub, bar, hotel", followed by the household. However, outbreaks of foodborne illness occurring in private homes are less likely to be reported than those in commercial and public premises, and it is believed that infections attributed to private homes are three times more frequent than those attributed to canteens (40). Food handlers also play an important role in the transmission of enteric viruses in the shellfish supply chain (16, 20, 41, 42), especially because shellfish specialities are generally prepared and served in restaurants. During production, harvest and packaging preparation, food can become contaminated with viruses by food handlers or after contact with virus-contaminated water and surfaces. A major contributor to the spread of disease in food production is poor hygiene practices or being in contact with faecal material or vomit (15). Food handlers are unaware of controls specific to enteric viruses (16). That is why training on all important aspects of NoVs and HAV according to the recently developed Codex Alimentarius guidelines to control viruses in food is strongly recommended. The primary purpose of the codex guidelines for the control of viruses in food is to give guidance on how to prevent or minimize the presence of human enteric viruses in food, especially NoVs and HAV, and to emphasize that management strategies regarding foodborne viruses and associated illnesses should be different from those for bacterial pathogens. In 2011 Poklar Vatovec with co-workers (42) carried out the research to evaluate the offer of shellfish specialities in Slovene Istria restaurants and to assess food safety knowledge and behaviour of food handlers in preparing shellfish dishes. Results indicated poor food safety knowledge regardless the education of food handlers. The origin of shellfish is important in ensuring food safety; and restaurants should be convinced of good raw meat to exclude foodborne poisoning. Therefore, shellfish should be bought only at registered plants, since these are under official supervision. However they observed that shellfish were not always bought at registered plants, but supplied from the so called illegal »black market«. Next to that it was also observed that employed personnel were hardly acquainted with HACCP principles which represent major food safety hazard. Cooking (at least 90 °C for at least 90 seconds) is a critical point for ensuring food safety. The survey showed that the mid temperature was measured by only 26.8% of the interviewees with formal education and 7.3% with informal education. The remaining did not perform this procedure or it was not known whether it was performed. The results of the survey demonstrated that only 4.9% of the interviewees, regardless their education, are familiar with the correct temperature for heat treatment of shellfish. Research also pointed out that food handlers employed in Slovene Istria restaurants have insufficient knowledge on storing temperatures, storing time and the adequate methods of storing shellfish. Pilot study: Semi-structured interview with Slovenian mussel farmers Pilot study illustration In order to determine eventual connection between comprehension of viral food safety and the shellfish growing practices, the four semi-structured interviews were carried out with Slovenian mussel farmers. The interview guide covered the following topics: - Factors responsible for food safety within the shellfish food supply chain, - Conditions related to food safety hazards with an emphasis on foodborne viruses due to virus-commodity combination, which has been identified as one of the greatest public health concerns. In this pilot study, an empirical grounding was important because an exploration of local farmers' viral food safety perceptions and their good hygiene practices, together with results from the National Zoonoses Monitoring Programme, outlined and gave insight into the current situation in Slovenia. The semi-structured interviews were chosen due to the sensitivity and complexity of the subject discussed. The semi-structured interview started with questions concerning food safety in general. The first open thematic question was: "Tell me as much as you can about the importance of the mussels production process, and about the factors that could affect its food safety." Discussions continued with the questions: "Could you please 200 M. Ambrozic, U. Henigman, M. Jevsnik, A. Kirbis, P. Raspor explain what food safety means from your point of view and when mussels are considered safe for a consumer?" and: "Have you ever heard about viruses that are transmitted by shellfish?" Follow-up questions were posed to complement and facilitate the dialogue. The discussions were concluded with the question: "Do you use working documents that have resulted from food safety legislation, like the HACCP plan, good practices that includes viruses?" The semi-structured interview ended with a question on whether the interviewee had something to add. Efforts were made to create trust, since issues of guilt and failure may easily arise. The results of national monitoring are supported by the responses recorded by the interviewers, which were clearly marked due to the assurance of anonymity. The letter "I" (I1) signifies "interview", while the number represents a running number of interviews. The semi-structured interviews with the local mussel farmers were qualitatively analyzed, using the grounded theory method. This theory produces theoretical models of individuals' perspectives of a given phenomenon and the strategies they use to resolve or cope with the problem in a distinct and bounded context (43, 44, 45). Interviews were recorded using a Dictaphone and later transcribed. Transcripts of the interviews were analyzed by coding the statements of the respondents using identified notions. These notions were gathered into topic categories (44, 45, 46). The validity is justified by triangulation. Three researchers with different disciplinary basic knowledge and different experiences in research were included in the analysis and interpretation process. Findings from semi-structured interviews Eight topics (Scheme 1) were identified following text analysis of the respondents' answers during semi-structured interviews: compliance with the legislation, transport, traceability, employer's awareness, food handlers' awareness and competencies, microbiological contamination, communal infrastructure, and disregarding an environmental code of conduct in the sea. These topics were obtained after identifying the topics in the statements. The interviewee's answers were very guarded; consequently, more comprehensive data processing cannot be achieved. With the intention to show comprehension within interviewee reflections, these identified topics (Scheme 1) were integrated with a specified section in Guidelines for the control of viruses in food ( 15) as follows: Primary production / Harvesting area; Establishment: Design and facilities; Control of operation; Establishment: Maintenance and sanitation; Establishment: Personal hygiene; Product information and consumer awareness; and Training. Specific topics (microbiological contamination, communal infrastructure, and disregarding an environmental code of conduct in the sea) represent an unrecognized threat to V-/ Primary production (microbiological ¡_ contamination of the waters; in which they grow) | Establishment: Design and ! facilities Control of operation | (cooking temperature versusr eating habits) V V Establishment: Maintenance!, and sanitation Personal hygiene ' Product information and . j consumer awarenenss (lot ! ; identification, labelling and [ | education) ; I Training (preventing faecal ! ! contamination, contagious > food handlers) Current recommendations CL O