survey of enteric virus presence in shellfish products in italy (1999-2008)

1 ICMSS09 – Nantes, France – June 2009

www.symposcience.org

Survey of Enteric Virus Presence In Shellfish Products In Italy (1999-2008) Croci Luciana1*, Losio Marina Nadia 2, Arcangeli Giuseppe 3, Pepe Tiziana 4, Pavoni Enrico 2, Magnabosco Cristian 3, Ventrone Iole 4, Suffredini Elisabetta 1 1 Istituto Superiore di Sanità, Dipartimento di Sanità Pubblica Veterinaria e Sicurezza Alimentare, v.le Regina Elena 299, 00161 Rome, Italy 2 Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, via Bianchi 7/9, 25124 Brescia, Italy 3 Istituto Zooprofilattico Sperimentale delle Venezie, via Leonardo da Vinci 39, 45011 Adria (RO), Italy 4 Università degli Studi di Napoli “Federico II”, Dipartimento di Scienze Zootecniche e Ispezione degli Alimenti, via Federico Delpino 1, 80137 Napoli, Italy

Enteric virus contamination in shellfish has been increasingly recognised as a risk for public health. In Italy, in the last decade, the surveillance of enteric viruses in seafood products has progressively joined the official controls required by European legislation. From 1999, after the first pilot study on Hepatitis A (HAV) in shellfish on the retail market in Puglia, the screening has been enlarged to Norovirus (NoV), Enterovirus (EV, year 2001) and Rotavirus (RV, 2003). In the period 1999-2008, with progressive improvements in the detection and characterization methods (conventional PCR, real-time PCR, sequencing, etc.), between 1205 (RV) and 2787 (HAV) samples were analysed, depending on the virus considered. Monitoring included shellfish at both the consumption level and production stage (sampling from class A and B harvesting areas in the Adriatic and Tyrrhenian Sea), nationally produced and imported products, products suspected for epidemics, and prepared and treated seafood products. Overall, at the retail level, enteric virus contamination involved approximately 5% of the samples (6.1% with HAV, 2.9% NoV, 5.2% EV and 0.9% RV), while higher contamination was detected in samples from class B harvesting areas. Genotypes most frequently detected were IA and IB for HAV and GII.4, GIIb, GII.1 and GII.2 for NoV. For cultivable viruses, presence of infective virus was confirmed for HAV in 29.0% and for EV in 12.9% of positive samples. These data provide a preliminary picture of the circulation level of different enteric viruses in seafood products. To evaluate the safety problem of viral

* Dott.ssa Luciana Croci Dipartimento di Sanità Pubblica Veterinaria e Sicurezza Alimentare Istituto Superiore di Sanità, Viale Regina Elena, 299 00161 Rome Italy fax +39 (0)6 49902045 e-mail: luciana.croci@iss.it

2 ICMSS09 – Nantes, France – June 2009

www.symposcience.org

contamination in shellfish, further systematic studies need to be carried out on harvesting areas using standardized methods and taking into account the environmental conditions influencing virus survival and spread.. Keywords: shellfish, enteric viruses, Hepatitis A virus, Norovirus, Enterovirus, Rotavirus, Italy

Introduction Bivalve shellfish are recognized as a potential vehicle of viral diseases (Lees 2000; Potasman et al. 2002). In accordance with the Reg. EC n. 1441 ⁄ 2007 (revising Reg. 2073 ⁄ 2005) the evaluation of microbiological quality of these products still relies on bacteriological contaminants (Salmonella and E. coli) and microbiological criteria for the enteric viruses have not been established because of the lack of a standard reference method. In the past decade, the molecular approach has led to the development of a large number of methods (Atmar et al. 1995; De Medici et al. 2004), particularly for the detection of viruses that, like HAV and NoV, grow poorly or not at all in cell culture (Duizer et al. 2004). The availability of such methods – despite the limitations due to the lack of international standardization – allowed knowledge to be gained on the incidence of enteric viruses associated with shellfish-related outbreaks (Gallimore et al. 2004; Prato et al. 2004; Sanchez et al. 2002) and their circulation in different areas (Formiga-Cruz et al. 2002; Le Guyader et al. 2000; Myrmel et al. 2004). In Italy, such investigations firstly (1999) focused mainly on detecting the hepatitis A virus in shellfish (De Medici et al. 2001a), on account of the high incidence of hepatitis A cases in some regions of southern Italy (http://www.iss.it/seie/index.php?lang=2). The screening has since been enlarged to Norovirus (NoV) (Croci et al. 2007; Prato et al. 2004; Suffredini et al. 2008), Enterovirus (EV, year 2001) and Rotavirus (RV, 2003). The aim of this paper is an evaluation of the presence and the distribution of enteric viruses (HAV, NoV, EV and RV) among the different shellfish species harvested and commercialised in Italy, by means of an overview of the results obtained by monitoring programs performed in various regions of Italy from 1999 to 2008.

1. Materials and methods

1.1. Samples From 1999 to 2008, multiple monitoring programs were performed for the evaluation of the presence of enteric viruses in shellfish (especially mussels, clams and oysters) in different production areas and markets of certain Italian regions (figure 1). The samples included shellfish at consumption level and production stage (sampling from class A and B harvesting areas in the Adriatic and Tyrrhenian sea), national and imported products, products suspected for epidemics, and prepared and treated seafood products.

The number of samples analysed varied between 1205 (RV) and 2787 (HAV) depending on the virus considered. The majority of samples represented products approved for direct consumption, while a fraction (n = 199) were collected from class B harvesting areas.

Figure 1: Areas involved in the monitoring programs Regions: 1: Lazio; 2: Emilia Romagna and Lombardia; 3: Puglia; 4: Campania; 5: Veneto

1.2. Methods Different methodologies were used according to the progressive improvements in the detection and characterization methods (conventional PCR, real-time PCR, sequencing, etc.). At the beginning (from 1999 to 2006) the whole shellfish body was analysed. Briefly, the homogenates (75 g) diluted 1:2 in glycine buffer were centrifuged (10000 x g for 15 min) and the viruses in the supernatant were concentrated through PEG precipitation (De Medici et al. 2001b); RNA extraction was performed according to Afzal and Minor (Afzal and Minor 1994) and conventional RT-PCR was used, depending on the virus, as follows:

- HAV: RT-nested-PCR or RT-seminested-PCR (De Medici et al. 2001b; Le Guyader et al. 1994) with positive samples confirmed by sequencing

- NoV: RT-booster-PCR with the results confirmed by Southern hybridization (De Medici et al. 2004) and sequencing (Croci et al. 2007)

- EV: RT-nested-PCR (Pina et al. 1998) - RV: RT-nested-PCR (Elschner et al. 2002).

For HAV and EV positive samples, virus infectivity was confirmed by inoculation of 1 ml mollusc extract respectively on Frp3 and BGM cell monolayers (De Medici et al. 2001b). The cultures, incubated at 37°C and 5%CO2 in Eagle minimum essential medium with 2% fetal bovine serum, were observed at regular intervals for 5 days (EV) or 15 days (HAV) until the appearance of a cytopathic effect. The propagation of the virus within the cells was confirmed by RT-PCR.

3 ICMSS09 – Nantes, France – June 2009

www.symposcience.org

4 ICMSS09 – Nantes, France – June 2009

www.symposcience.org

In the more recent years (2007-2008), to increase recovery and reduce PCR inhibitors (Comelli et al. 2008) virus extraction was performed on digestive gland with proteinase K treatment (Jothikumar et al. 2005) and RNA was extracted and purified with a NucliSens extraction kit (bioMerieux). In the same period, the molecular detection procedure described for HAV and NoV was replaced by real-time RT-PCR (Costafreda et al. 2006; da Silva et al. 2007). During the period of this study, epidemiological data on HAV infection in the population were collected by the Italian National Surveillance System for Acute Viral Hepatitis (SEIEVA - http://www.iss.it/seie/index.php?lang=2).

2. Results Table 1 shows the total number of hepatitis A cases verified in Italy from 1999-2008, the percentage of these cases attributed to shellfish consumption and the number of the shellfish samples analysed and positive for HAV presence. The percentage of HAV-positive samples varied between 0.9 to 14.1%, depending on the region monitored. The infectivity test, when performed, confirmed the presence of infectious HAV in a minority of the positive samples (29.0%) and sequencing showed the prevalence of genotype IA among circulating strains.

Table 1: Number of Hepatitis A cases and shellfish samples analysed (1999-2008)

Cases Shellfish samples analysed Year

total shellfish-related % total positive % infectious

Sampling areas

1999 794 440 58 170 24 14.1 n.a. 1

2000 934 597 68 365 32 8.8 n.a. 2

2001 970 626 73 565 53 9.4 18 2 and 3

2002 711 434 65 137 8 5.8 n.a. 2

2003 1013 592 63 199 10 5.0 0 2

2004 1178 unk 411 25 6.1 11 2 and 4

2005 678 unk 346 3 0.9 1 2 and 5

2006 490 214 44 208 0 0.0 2 and 5

2007 716 533 76 182 10 5.5 0 2

2008 n.a. n.a.

204 6 2.9 1 2 and 4

total 2787 171 6.1 unk: unknown n.a.: not available Genotyping, when performed, showed the presence of genotypes IA and IB Table 2 gives the results for NoV detection. In this case, the highest percentages were obtained in 2003 and 2008 and were related to the monitoring of harvesting areas.

5 ICMSS09 – Nantes, France – June 2009

www.symposcience.org

Sequencing showed that the circulating genotypes were GII.4, GIIb, GII.1 and GII.2 while GI genogroup was detected only in the later years when the application of real-time RT-PCR increased the sensitivity of the detection method (Suffredini et al. 2008). Table 3 gives the results obtained for the other viruses considered. EV in some years (2003 and 2007) were detected in a high percentage of samples, respectively 13.6 and 16.1%, but infectivity, when tested, was only confirmed in a limited number of cases; RV were usually present at a very low frequency. Overall, contamination by enteric viruses involved approximately 5% of the shellfish samples analysed from 1999 to 2008; in particular, 6.1% were contaminated by HAV, 5.8% by NoV, 6.5% by EV and 0.9% by RV. In the subset of samples from the retail level and class A areas (which apply the same microbiological criteria) the viral contamination involved 6.1% of samples for HAV, 2.9% for NoV, 5.2% for EV and 0.9% for RV. The analyses of shellfish from class B areas (n = 199) provided 31.1% of positive samples for NoV and 3.0% of positives for HAV. Among the different shellfish species analysed, results showed that clams were particularly concerned by viral contamination, with 11.2% positive samples compared with 8.1% of mussels and 2.1% of oysters (table 4).

Table 2: Norovirus contamination in shellfish (2001-2008)

Shellfish samples analysed Sampling areas Genotypes Year

total positive % 2001 50 0 0.0 1 2002 137 0 0.0 1 2003 199 32 16.1 1 GII.1; GII.2; GII.4 2004 274 9 3.3 1 GIIb 2005 359 4 1.1 1 and 2 GIIb 2006 202 7 3.5 1 and 2 GII.4; GIIb 2007 170 1 0.6 1 GIIb 2008 206 39 18.9 1 and 3 GII.4; GII.4 var2006b;

GII.1; GII.2; GIIb

total 1597 92 5.8

Table 3: Enterovirus and Rotavirus contamination in shellfish (2001-2008) Enterovirus: shellfish

samples analysed Rotavirus: shellfish samples analysed Year

total positive % infectious total positive %

Sampling areas

2001 150 4 2.7 1 2002 137 1 0.7 1 2003 199 27 13.6 137 0 0.0 1 2004 273 22 8.1 4 265 4 1.5 1 2005 325 9 2.8 0 325 3 0.9 1 and 2 2006 202 12 5.9 1 202 1 0.5 1 and 2 2007 168 27 16.1 4 168 2 1.2 1 2008 193 5 2.6 108 1 0.9 1 total 1647 107 6.5 1205 11 0.9

6 ICMSS09 – Nantes, France – June 2009

www.symposcience.org

Table 4: Hepatitis A and Norovirus contamination in different shellfish species analysed (1999-2008)

Matrix Samples Positive (HAV and/or NoV) %

Mussels 1893 154 8.1 Clams 685 77 11.2 Oysters 141 3 2.1

Processed* 68 2 2.9

total 2787 236 8.5 * shelled mussels, packaged seafood products subjected to heat treatment, etc.

Conclusions Viral contamination, which appeared at different levels according to the virus considered, affected shellfish collected both from harvesting areas (A and B) and at the retail level. Class B harvesting areas were particularly affected by NoV contamination (31.1% of contaminated samples), while the lower percentage detected in shellfish collected from markets (2.9%) could be related to the depuration process applied to these samples (De Medici et al. 2001a; Suffredini et al. 2008). Even if depuration is not adequate to completely remove the virus present, it could reduce its concentration to levels not detectable by the analytical methods. On the other hand, the percentage of the HAV-contaminated samples was higher at the retail level (6.1%) than in shellfish from class B harvesting areas (3.0%) and this could be due both to imported products and to the forbidden, though common, practice of local retailers of storing shellfish in uncontrolled seawater (Pontrelli et al. 2008). Among the different shellfish species, viral contamination seemed more frequent in clams than in the other examined species and this difference may be related to the fact that the growing areas for clams are generally nearer to the coast than those used for mussels. For cultivable viruses, infectivity - when tested - was confirmed in only 29.0% of HAV- and 12.9% of EV-positive samples, confirming that nucleic acid detection and presence of infectious viruses are not always associated. This should be taken into account in the evaluation of the risk for consumers once molecular methods are in use for the routine control of seafood. Additionally, as highlighted by the influence of real-time RT-PCR adoption on NoV GI detection in this study, the applied molecular methods should be constantly evaluated and, where necessary, revised in accordance with technical progress. . The present data, although not representative of the total situation and limited by non homogenous sampling plans and different analytical methods, provide a preliminary picture on the circulation of enteric viruses in seafood products in Italy Further resources should be devoted to developing appropriate epidemiological surveillance and monitoring plans (taking into account specific characteristics of the areas, atmospheric events and other environmental aspects) to understand how the viruses spread within the population and to know the prevalence of enteric viruses and the circulation of different strains. These actions will provide data for risk assessment, improvement of the

Commentaire [HM1] : insert ‘human’ here?

7 ICMSS09 – Nantes, France – June 2009

www.symposcience.org

safety of seafood products and planning of more effective informative campaigns to heighten consumer awareness.

References Afzal M.A., Minor P.D., 1994. Instant RNA isolation from virus-infected tissue culture fluid for the

polymerase chain reaction, Vaccine, 12, Aug, p. 976-977. Atmar R.L., Neill F.H., Romalde J.L., Le Guyader F., Woodley C.M., Metcalf T.G.,Estes M.K.,

1995. Detection of Norwalk virus and hepatitis A virus in shellfish tissues with the PCR, Applied and Environmental Microbiology, 61, Aug, p. 3014-3018.

Comelli H.L., Rimstad E., Larsen S.,Myrmel M., 2008. Detection of norovirus genotype I.3b and II.4 in bioaccumulated blue mussels using different virus recovery methods, International Journal of Food Microbiology, 127, Sep 30, p. 53-59.

Costafreda M.I., Bosch A., Pinto R.M., 2006. Development, evaluation, and standardization of a real-time TaqMan reverse transcription-PCR assay for quantification of hepatitis A virus in clinical and shellfish samples, Applied and Environmental Microbiology, 72, Jun, p. 3846-3855.

Croci L., Losio M.N., Suffredini E., Pavoni E., Di Pasquale S., Fallacara F.,Arcangeli G., 2007. Assessment of human enteric viruses in shellfish from the northern Adriatic sea, International Journal of Food Microbiology, 114, Mar 10, p. 252-257.

da Silva A.K., Le Saux J.C., Parnaudeau S., Pommepuy M., Elimelech M.,Le Guyader F.S., 2007. Evaluation of removal of noroviruses during wastewater treatment, using real-time reverse transcription-PCR: different behaviors of genogroups I and II, Applied and Environmental Microbiology, 73, Dec, p. 7891-7897.

De Medici D., Ciccozzi M., Fiore A., Di Pasquale S., Parlato A., Ricci-Bitti P.,Croci L., 2001a. Closed-circuit system for the depuration of mussels experimentally contaminated with hepatitis A virus, Journal of Food Protection, 64, Jun, p. 877-880.

De Medici D., Croci L., Di Pasquale S., Fiore A.,Toti L., 2001b. Detecting the presence of infectious hepatitis A virus in molluscs positive to RT-nested-PCR, Letters in Applied Microbiology, 33, Nov, p. 362-366.

De Medici D., Croci L., Suffredini E.,Toti L., 2004. Reverse transcription-booster PCR for detection of noroviruses in shellfish, Applied and Environmental Microbiology, 70, Oct, p. 6329-6332.

Duizer E., Schwab K.J., Neill F.H., Atmar R.L., Koopmans M.P.,Estes M.K., 2004. Laboratory efforts to cultivate noroviruses, Journal of General Virology, 85, Jan, p. 79-87.

Elschner M., Prudlo J., Hotzel H., Otto P.,Sachse K., 2002. Nested reverse transcriptase-polymerase chain reaction for the detection of group A rotaviruses, Journal of veterinary medicine. B, Infectious diseases and veterinary public health, 49, Mar, p. 77-81.

Formiga-Cruz M., Tofino-Quesada G., Bofill-Mas S., Lees D.N., Henshilwood K., Allard A.K., Conden-Hansson A.C., Hernroth B.E., Vantarakis A., Tsibouxi A., Papapetropoulou M., Furones M.D.,Girones R., 2002. Distribution of human virus contamination in shellfish from different growing areas in Greece, Spain, Sweden, and the United Kingdom, Applied and Environmental Microbiology, 68, Dec, p. 5990-5998.

Gallimore C.I., Green J., Richards A.F., Cotterill H., Curry A., Brown D.W.,Gray J.J., 2004. Methods for the detection and characterisation of noroviruses associated with outbreaks of gastroenteritis: outbreaks occurring in the north-west of England during two norovirus seasons, Journal of Medical Virology, 73, Jun, p. 280-288.

Jothikumar N., Lowther J.A., Henshilwood K., Lees D.N., Hill V.R.,Vinje J., 2005. Rapid and sensitive detection of noroviruses by using TaqMan-based one-step reverse transcription-PCR assays and application to naturally contaminated shellfish samples, Applied and Environmental Microbiology, 71, Apr, p. 1870-1875.

Le Guyader F., Dubois E., Menard D.,Pommepuy M., 1994. Detection of hepatitis A virus, rotavirus, and enterovirus in naturally contaminated shellfish and sediment by reverse transcription-seminested PCR, Applied and Environmental Microbiology, 60, Oct, p. 3665-3671.

8 ICMSS09 – Nantes, France – June 2009

www.symposcience.org

Le Guyader F., Haugarreau L., Miossec L., Dubois E.,Pommepuy M., 2000. Three-year study to assess human enteric viruses in shellfish, Applied and Environmental Microbiology, 66, Aug, p. 3241-3248.

Lees D., 2000. Viruses and bivalve shellfish, International Journal of Food Microbiology, 59, Jul 25, p. 81-116.

Myrmel M., Berg E.M., Rimstad E.,Grinde B., 2004. Detection of enteric viruses in shellfish from the Norwegian coast, Applied and Environmental Microbiology, 70, May, p. 2678-2684.

Pina S., Puig M., Lucena F., Jofre J.,Girones R., 1998. Viral pollution in the environment and in shellfish: human adenovirus detection by PCR as an index of human viruses, Applied and Environmental Microbiology, 64, Sep, p. 3376-3382.

Pontrelli G., Boccia D., M D.I.R., Massari M., Giugliano F., Celentano L.P., Taffon S., Genovese D., S D.I.P., Scalise F., Rapicetta M., Croci L.,Salmaso S., 2008. Epidemiological and virological characterization of a large community-wide outbreak of hepatitis A in southern Italy, Epidemiology and Infection, 136, Aug, p. 1027-1034.

Potasman I., Paz A.,Odeh M., 2002. Infectious outbreaks associated with bivalve shellfish consumption: a worldwide perspective, Clinical Infectious Diseases, 35, Oct 15, p. 921-928.

Prato R., Lopalco P.L., Chironna M., Barbuti G., Germinario C.,Quarto M., 2004. Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in south Italy, BMC Infectious Diseases, 4, Sep 21, p. 37.

Sanchez G., Pinto R.M., Vanaclocha H.,Bosch A., 2002. Molecular characterization of hepatitis a virus isolates from a transcontinental shellfish-borne outbreak, Journal of Clinical Microbiology, 40, Nov, p. 4148-4155.

Suffredini E., Corrain C., Arcangeli G., Fasolato L., Manfrin A., Rossetti E., Biazzi E., Mioni R., Pavoni E., Losio M.N., Sanavio G.,Croci L., 2008. Occurrence of enteric viruses in shellfish and relation to climatic-environmental factors, Letters in Applied Microbiology, 47, Nov, p. 467-474.