Elisabetta Suffredini

Detection of Multiple Noroviruses Associated with an International Gastroenteritis Outbreak Linked to Oyster Consumption

ABSTRACT An international outbreak linked to oyster consumption involving a group of over 200 people in Italy and 127 total subjects in 13 smaller clusters in France was analyzed using epidemiological and clinical data and shellfish samples. Environmental information from the oyster-producing area, located in a lagoon in southern France, was collected to investigate the possible events leading to the contamination. Virologic analyses were conducted by reverse transcription-PCR (RT-PCR) using the same primer sets for both clinical and environmental samples. After sequencing, the data were analyzed through the database operated by the scientific network FoodBorne Viruses in Europe. The existence of an international collaboration between laboratories was critical to rapidly connect the data and to fully interpret the results, since it was not obvious that one food could be the link because of the diversity of the several norovirus strains involved in the different cases. It was also demonstrated that heavy rain was responsible for the accidental contamination of seafood, leading to a concentration of up to hundreds of genomic copies per oyster as detected by real-time RT-PCR.

Effects of depuration of molluscs experimentally contaminated with Escherichia coli, Vibrio cholerae O1 and Vibrio parahaemolyticus

Aims: The aim of the present study was to investigate the behaviour of two pathogenic vibrios (Vibrio cholerae O1 and Vibrio parahaemolyticus) during depuration and to compare it with that of Escherichia coli, used as an indicator of suitability for consumption.

Comparison of different biochemical and molecular methods for the identification of Vibrio parahaemolyticus

Aims:  Multicentre evaluation of biochemical and molecular methods for the identification of Vibrio parahaemolyticus.

Detection of Vibrionaceae in mussels and in their seawater growing area

L. CROCI, P. SERRATORE, L. COZZI, A. STACCHINI, S. MILANDRI, E. SUFFREDINI AND L. TOTI. 2001.

Occurrence of enteric viruses in shellfish and relation to climatic-environmental factors

Aims:  To investigate the presence of enteric viruses [hepatitis A (HAV) and norovirus (NoV)] in shellfish harvested from the deltaic area of the Po river in relation to environmental factors.

Qualitative and quantitative assessment of viral contamination in bivalve molluscs harvested in Italy.

Bivalve molluscs are a well documented source of viral infection. Further data on shellfish viral contamination are needed to implement European Regulations with sanitary measures more effective against viral pathogens. To this aim, 336 samples of bivalve molluscs (185 mussels, 66 clams, 23 oysters and 62 samples from other species) collected in harvesting areas of class A and B of four Italian Regions were analyzed for qualitative and quantitative determination of hepatitis A virus (HAV) and Norovirus (NoV) GI and GII, using real time RT-PCR. The results showed a wide diffusion of viral contamination in the shellfish production areas considered. HAV prevalence was low (0.9%) with contamination levels that varied from 5 to 7 × 10(2)copies/g. On the contrary, NoV showed a high prevalence (51.5%), with a large variability according to the group considered (e.g. 47.8% for Crassostrea in Veneto, 79.7% for Mytilus in Campania, 84.6% for Tapes in Sardinia). NoV contamination affected class A and class B production areas to a different extent, with a statistically significant difference in both contamination prevalence (22.1% vs. 66.3%; p<0.0001) and quantity (average contamination level of 3.1 × 10(2) vs. 1.9 × 10(3) copies/g; p<0.05). The different species analyzed from class B harvesting areas (Mytilus, Tapes/Ruditapes and Crassostrea) showed a NoV prevalence respectively of 70.3%, 66.0% and 47.8% but comparable NoV contamination levels (between 8.4 × 10(2) and 4.9 × 10(3)copies/g). Other two bivalve species considered in the study (Donax spp. and Solen spp.) showed a relevant NoV presence (40.0% and 34.4% of samples). Finally, samples analyzed before and after commercial purification treatment showed a decrease of contamination prevalence after the treatment, but inconsistent results were recorded on NoV levels. The data obtained, together with other quantitative information to estimate consumer exposure, in association with studies on dose-response and on the effectiveness of post-harvest treatments, will provide a useful tool for the definition of microbiological criteria related to the different shellfish species.

Reverse transcription-booster PCR for detection of noroviruses in shellfish.

ABSTRACT The methods commonly used for norovirus (NV) detection are based on reverse transcription-PCR (RT-PCR) followed by confirmation of the amplified sequence. To increase sensitivity, an RT-booster PCR was developed. The proposed method showed an increase in sensitivity at least 2 log units for all the NV strains tested compared with the standard RT-PCR method. Higher sensitivity was confirmed in tests on experimentally and naturally contaminated shellfish.

Norovirus contamination in different shellfish species harvested in the same production areas

To investigate Norovirus (NoV) contamination of mussels, clams and oysters harvested in two class B harvesting areas of the delta of the Po river, to choose a species as an indicator.

Detection and quantification of Vibrio parahaemolyticus in shellfish from Italian production areas.

Vibrio parahaemolyticus is a marine microorganism, recognized as an important cause of foodborne illness particularly in Asia, South America and United States. Outbreaks are rarely reported in Europe, but they can occur unexpectedly in relation, among other reasons, to the spread of highly virulent strains. It is known that the risk is proportional to exposure levels to pathogenic V. parahaemolyticus (i.e. carrying the tdh and/or the trh genes) but currently there is a lack of occurrence data for pathogenic V. parahaemolyticus in shellfish production areas of the Member States. In this study a total of 147 samples of bivalve molluscs, from harvesting areas of two Italian regions (Sardinia and Veneto) were analyzed for Escherichia coli and salmonella, according to Reg 2073/2005, and for detection and enumeration of total and toxigenic V. parahaemolyticus strains using a new DNA colony hybridization method. Environmental parameters (water temperature and salinity) were also recorded. Results of E. coli were consistently in agreement with the legislation limits for the harvesting class of origin and Salmonella was detected only in one sample. The average contamination levels for total V. parahaemolyticus were 84 and 73 CFU/g respectively for Sardinia and Veneto, with the highest value reaching 8.7 × 10(3)CFU/g. Nineteen samples (12.9%) resulted positive for the presence of potentially pathogenic V. parahaemolyticus strains, with levels ranging between 10 and 120 CFU/g and most of the positive samples (n=17) showing values equal or below 20 CFU/g. A significant correlation (r=0.41) was found between water temperature and V. parahaemolyticus levels, as well as with isolation frequency. The data provided in this study on contamination levels of total and potentially pathogenic V. parahaemolyticus, seasonal distribution and correlation with water temperature, will help in defining appropriate monitoring programs and post-harvest policies for this hazard, improving the management of the harvesting areas and the safety of bivalve molluscs.

Noroviruses in Seafood: A 9-Year Monitoring in Italy

Norovirus (NoV) are increasingly important as etiological agents of gastrointestinal infections. Consumption of bivalve molluscs and ready-to-eat fishery products is one of the most common ways of acquiring NoV foodborne infections, and the rise of outbreaks of viral gastroenteritis represents an important health problem that is also responsible for economic losses. The aim of this work was to define the prevalence of NoV contamination in preserved fishery products and in shellfish commercialized in Italy, taking into account the results obtained during 9 years of survey (2003-2011) and paying special attention to the regions more involved in national production. A total of 4463 samples were examined (2310 mussels, 1517 clams, 510 oysters, 22 other shellfish species, 104 preserved seafood products) and the average positivity rate for NoV presence was 4.1% and ranged from 0.6% in 2007 to 9.8% in 2003 and from 1.9% in preserved seafood products to 4.7% in mussels. Genetic characterization of circulating strains showed a prevalence of genogroup II genotypes, including GII.b and GII.e polymerase types and different GII.4 variants. This information could contribute to the optimization of risk-based sampling strategies for NoV contamination in seafood, taking into account variability in different species and from year to year.