Evaluation of potential indicators of viral contamination in shellfish and their applicability to diverse geographical areas

The distribution of the concentration of potential indicators of fecal viral pollution in shellfish was analyzed under diverse conditions over 18 months in diverse geographical areas. These microorganisms have been evaluated in relation to contamination by human viral pathogens detected in parallel in the analyzed shellfish samples. Thus, significant shellfish-growing areas from diverse countries in the north and south of Europe (Greece, Spain, Sweden, and the United Kingdom) were defined and studied by analyzing different physicochemical parameters in the water and the levels of Escherichia coli, F-specific RNA bacteriophages, and phages infecting Bacteroides fragilis strain RYC2056 in the shellfish produced, before and after depuration treatments. A total of 475 shellfish samples were studied, and the results were statistically analyzed. According to statistical analysis, the presence of human viruses seems to be related to the presence of all potential indicators in the heavily contaminated areas, where E. coli would probably be suitable as a fecal indicator. The F-RNA phages, which are present in higher numbers in Northern Europe, seem to be significantly related to the presence of viral contamination in shellfish, with a very weak predictive value for hepatitis A virus, human adenovirus, and enterovirus and a stronger one for Norwalk-like virus. However, it is important to note that shellfish produced in A or clean B areas can sporadically contain human viruses even in the absence of E. coli or F-RNA phages. The data presented here will be useful in defining microbiological parameters for improving the sanitary control of shellfish consumed raw or barely cooked.     

Evaluation of Potential Indicators of Viral Contamination in Shellfish and Their Applicability to Diverse Geographical Areas

The distribution of the concentration of potential indicators of fecal viral pollution in shellfish was analyzed under diverse conditions over 18 months in diverse geographical areas. These microorganisms have been evaluated in relation to contamination by human viral pathogens detected in parallel in the analyzed shellfish samples. Thus, significant shellfish-growing areas from diverse countries in the north and south of Europe (Greece, Spain, Sweden, and the United Kingdom) were defined and studied by analyzing different physicochemical parameters in the water and the levels of Escherichia coli, F-specific RNA bacteriophages, and phages infecting Bacteroides fragilis strain RYC2056 in the shellfish produced, before and after depuration treatments. A total of 475 shellfish samples were studied, and the results were statistically analyzed. According to statistical analysis, the presence of human viruses seems to be related to the presence of all potential indicators in the heavily contaminated areas, where E. coli would probably be suitable as a fecal indicator. The F-RNA phages, which are present in higher numbers in Northern Europe, seem to be significantly related to the presence of viral contamination in shellfish, with a very weak predictive value for hepatitis A virus, human adenovirus, and enterovirus and a stronger one for Norwalk-like virus. However, it is important to note that shellfish produced in A or clean B areas can sporadically contain human viruses even in the absence of E. coli or F-RNA phages. The data presented here will be useful in defining microbiological parameters for improving the sanitary control of shellfish consumed raw or barely cooked.     

Environmental factors influencing human viral pathogens and their potential indicator organisms in the blue mussel,Mytilus edulis: the first Scandinavian report

This study was carried out in order to investigate human enteric virus contaminants in mussels from three sites on the west coast of Sweden, representing a gradient of anthropogenic influence. Mussels were sampled monthly during the period from February 2000 to July 2001 and analyzed for adeno-, entero-, Norwalk-like, and hepatitis A viruses as well as the potential viral indicator organisms somatic coliphages, F-specific RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, and Escherichia coli. The influence of environmental factors such as water temperature, salinity, and land runoff on the occurrence of these microbes was also included in this study. Enteric viruses were found in 50 to 60% of the mussel samples, and there were no pronounced differences between the samples from the three sites. E. coli counts exceeded the limit for category A for shellfish sanitary safety in 40% of the samples from the sites situated in fjords. However, at the site in the outer archipelago, this limit was exceeded only once, in March 2001, when extremely high levels of atypical indole-negative strains of E. coli were registered at all three sites. The environmental factors influenced the occurrence of viruses and phages differently, and therefore, it was hard to find a coexistence between them. This study shows that, for risk assessment, separate modeling should be done for every specific area, with special emphasis on environmental factors such as temperature and land runoff. The present standard for human fecal contamination, E. coli, seems to be an acceptable indicator of only local sanitary contamination; it is not a reliable indicator of viral contaminants in mussels. To protect consumers and get verification of "clean" mussels, it seems necessary to analyze for viruses as well. The use of a molecular index of the human contamination of Swedish shellfish underscores the need for reference laboratories with high-technology facilities.     

Environmental Factors Influencing Human Viral Pathogens and Their Potential Indicator Organisms in the Blue Mussel, Mytilus edulis: the First Scandinavian Report

This study was carried out in order to investigate human enteric virus contaminants in mussels from three sites on the west coast of Sweden, representing a gradient of anthropogenic influence. Mussels were sampled monthly during the period from February 2000 to July 2001 and analyzed for adeno-, entero-, Norwalk-like, and hepatitis A viruses as well as the potential viral indicator organisms somatic coliphages, F-specific RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, and Escherichia coli. The influence of environmental factors such as water temperature, salinity, and land runoff on the occurrence of these microbes was also included in this study. Enteric viruses were found in 50 to 60% of the mussel samples, and there were no pronounced differences between the samples from the three sites. E. coli counts exceeded the limit for category A for shellfish sanitary safety in 40% of the samples from the sites situated in fjords. However, at the site in the outer archipelago, this limit was exceeded only once, in March 2001, when extremely high levels of atypical indole-negative strains of E. coli were registered at all three sites. The environmental factors influenced the occurrence of viruses and phages differently, and therefore, it was hard to find a coexistence between them. This study shows that, for risk assessment, separate modeling should be done for every specific area, with special emphasis on environmental factors such as temperature and land runoff. The present standard for human fecal contamination, E. coli, seems to be an acceptable indicator of only local sanitary contamination; it is not a reliable indicator of viral contaminants in mussels. To protect consumers and get verification of “clean” mussels, it seems necessary to analyze for viruses as well. The use of a molecular index of the human contamination of Swedish shellfish underscores the need for reference laboratories with high-technology facilities.     

Artificial neural network prediction of viruses in shellfish

A database was probed with artificial neural network (ANN) and multivariate logistic regression (MLR) models to investigate the efficacy of predicting PCR-identified human adenovirus (ADV), Norwalk-like virus (NLV), and enterovirus (EV) presence or absence in shellfish harvested from diverse countries in Europe (Spain, Sweden, Greece, and the United Kingdom). The relative importance of numerical and heuristic input variables to the ANN model for each country and for the combined data was analyzed with a newly defined relative strength effect, which illuminated the importance of bacteriophages as potential viral indicators. The results of this analysis showed that ANN models predicted all types of viral presence and absence in shellfish with better precision than MLR models for a multicountry database. For overall presence/absence classification accuracy, ANN modeling had a performance rate of 95.9%, 98.9%, and 95.7% versus 60.5%, 75.0%, and 64.6% for the MLR for ADV, NLV, and EV, respectively. The selectivity (prediction of viral negatives) was greater than the sensitivity (prediction of viral positives) for both models and with all virus types, with the ANN model performing with greater sensitivity than the MLR. ANN models were able to illuminate site-specific relationships between microbial indicators chosen as model inputs and human virus presence. A validation study on ADV demonstrated that the MLR and ANN models differed in sensitivity and selectivity, with the ANN model correctly identifying ADV presence with greater precision.     

Bioaccumulation Efficiency,Tissue Distribution,and Environmental Occurrence of Hepatitis E Virus in Bivalve Shellfish from France

Hepatitis E virus (HEV), an enteric pathogen of both humans and animals, is excreted by infected individuals and is therefore present in wastewaters and coastal waters. As bivalve molluscan shellfish are known to concentrate viral particles during the process of filter feeding, they may accumulate this virus. The bioaccumulation efficiencies of oysters (Crassostrea gigas), flat oysters (Ostrea edulis), mussels (Mytilus edulis), and clams (Ruditapes philippinarum) were compared at different time points during the year. Tissue distribution analysis showed that most of the viruses were concentrated in the digestive tissues of the four species. Mussels and clams were found to be more sensitive to sporadic contamination events, as demonstrated by rapid bioaccumulation in less than 1 h compared to species of oysters. For oysters, concentrations increased during the 24-h bioaccumulation period. Additionally, to evaluate environmental occurrence of HEV in shellfish, an environmental investigation was undertaken at sites potentially impacted by pigs, wild boars, and human waste. Of the 286 samples collected, none were contaminated with hepatitis E virus, despite evidence that this virus is circulating in some French areas. It is possible that the number of hepatitis E viral particles discharged into the environment is too low to detect or that the virus may have a very short period of persistence in pig manure and human waste.     

Virus-Contaminated Oysters: a Three-Month Monitoring of Oysters Imported to Switzerland

Molluscan shellfish are known to be carriers of viral and bacterial pathogens. The consumption of raw oysters has been repeatedly linked to outbreaks of viral gastroenteritis and hepatitis A. Switzerland imports over 300 tons of oysters per year, 95% of which originate in France. To assess the level of viral contamination, a 3-month monitoring study was conducted. Therefore, the sensitivities of several previously described methods for virus concentration were compared, and one protocol was finally chosen by using dissected digestive tissues. Eighty-seven samples consisting of five oysters each were analyzed for Norwalk-like viruses (NLVs), enteroviruses, and hepatitis A viruses from November 2001 to February 2002. The oysters were exported by 31 French, three Dutch, and two Irish suppliers. Eight oyster samples from six French suppliers were positive for NLVs, and four samples from four French suppliers were positive for enteroviruses; two of the latter samples were positive for both viral agents. No hepatitis A viruses were detected. The sequences of NLV and enterovirus amplicons showed a great variety of strains, especially for the NLVs (strains similar to Bristol, Hawaii, Mexico, and Melksham agent). The data obtained indicated that imported oysters might be a source of NLV infection in Switzerland. However, further studies are needed to determine the quantitative significance of the risk factor within the overall epidemiology of NLVs.     

Virus-contaminated oysters: a three-month monitoring of oysters imported to Switzerland

Molluscan shellfish are known to be carriers of viral and bacterial pathogens. The consumption of raw oysters has been repeatedly linked to outbreaks of viral gastroenteritis and hepatitis A. Switzerland imports over 300 tons of oysters per year, 95% of which originate in France. To assess the level of viral contamination, a 3-month monitoring study was conducted. Therefore, the sensitivities of several previously described methods for virus concentration were compared, and one protocol was finally chosen by using dissected digestive tissues. Eighty-seven samples consisting of five oysters each were analyzed for Norwalk-like viruses (NLVs), enteroviruses, and hepatitis A viruses from November 2001 to February 2002. The oysters were exported by 31 French, three Dutch, and two Irish suppliers. Eight oyster samples from six French suppliers were positive for NLVs, and four samples from four French suppliers were positive for enteroviruses; two of the latter samples were positive for both viral agents. No hepatitis A viruses were detected. The sequences of NLV and enterovirus amplicons showed a great variety of strains, especially for the NLVs (strains similar to Bristol, Hawaii, Mexico, and Melksham agent). The data obtained indicated that imported oysters might be a source of NLV infection in Switzerland. However, further studies are needed to determine the quantitative significance of the risk factor within the overall epidemiology of NLVs.     

Ionic bonding, the mechanism of viral uptake by shellfish mucus.

An investigation was conducted to determine the processes involved in the contamination of shellfish by viruses. Results of binding-release studies show that the process involves the attachment of viruses to mucus secreted, and then ingested, by shellfish during feeding. Analysis of the mucus-virus bond involved selective degradation of the mucus and use of chemical agents to block carboxyl and sulfate groups on the mucus. Results obtained indicate that the attachment of virus to mucus is primarily ionic and involves the binding of viral particles to sulfate radicals on the mucopolysaccharide moiety of shellfish mucus.     

Ionic bonding, the mechanism of viral uptake by shellfish mucus.

An investigation was conducted to determine the processes involved in the contamination of shellfish by viruses. Results of binding-release studies show that the process involves the attachment of viruses to mucus secreted, and then ingested, by shellfish during feeding. Analysis of the mucus-virus bond involved selective degradation of the mucus and use of chemical agents to block carboxyl and sulfate groups on the mucus. Results obtained indicate that the attachment of virus to mucus is primarily ionic and involves the binding of viral particles to sulfate radicals on the mucopolysaccharide moiety of shellfish mucus.     

Molecular Detection and Characterization of Gastroenteritis Viruses Occurring Naturally in the Stream Waters of Manaus, Central Amazônia, Brazil

To assess the presence of the four main viruses responsible for human acute gastroenteritis in a hydrographic network impacted by a disordered urbanization process, a 1-year study was performed involving water sample collection from streams in the hydrographic basin surrounding the city of Manaus, Amazonas, Brazil. Thirteen surface water sample collection sites, including different areas of human settlement characterized as urban, rural, and primary forest, located in the Tarumã-Açu, São Raimundo, Educandos, and Puraquequara microbasins, were defined with a global positioning system. At least one virus was detected in 59.6% (31/52) of the water samples analyzed, and rotavirus was the most frequent (44.2%), followed by human adenovirus (30.8%), human astrovirus (15.4%), and norovirus (5.8%). The viral contamination observed mainly in the urban streams reflected the presence of a local high-density population and indicated the gastroenteritis burden from pathogenic viruses in the water, principally due to recreational activities such as bathing. The presence of viral genomes in areas where fecal contamination was not demonstrated by bacterial indicators suggests prolonged virus persistence in aquatic environments and emphasizes the enteric virus group as the most reliable for environmental monitoring.     

Seasonal detection of human viruses and coliphage in Newport Bay, California

Recent studies have shown that the fecal indicator bacteria (FIB) currently used to indicate water quality in the coastal environment may be inadequate to reflect human viral contamination. Coliphage was suggested as a better indicator of human viral pollution and was proposed by the U.S. EPA as an alternative indicator for fecal pollution in groundwater. In this study, we investigated the occurrence and distribution of FIB, F+ coliphage, and PCR-detectable human adenovirus and enterovirus for an entire year at 15 locations around the Newport Bay watershed, an important southern California estuary for water recreation and an ecological reserve. Peak concentrations and prevalences of FIB and F+ coliphage were associated with winter storms (wet weather). Human adenoviruses and enteroviruses, however, were detected by PCR in approximately 5% of samples collected in the summer (dry weather) but only once in wet weather. These results demonstrated that FIB and coliphage have similar seasonal and freshwater-to-saltwater distribution patterns, while the detection of human viruses depends on a distribution pattern that is the opposite of that of FIB and coliphage. This research suggested that coliphage and FIB share similar environmental sources, while sources of human viruses in Newport Bay are perhaps different.     

Assessment of enterovirus contamination in mussel samples from Morocco

Summary In Morocco, shellfish sanitary quality analysis does not currently include enteric virus detection. Enteroviruses are classically detected by cell culture, but molecular methods such as RT-PCR are now broadly used alone or associated to cell culture. RT-PCR has the advantage of requiring less time and budget than cell culture. Bivalve mussels, being filter-feeders tend to accumulate viruses in contaminated seawater. In order to assess mussel contamination by enteroviruses, we screened samples of two origins, an aquaculture system and an area where wild mussels grow. Domestic sewage samples from an outlet near the above wild mussels growing area were also analysed. Viruses were concentrated from mussel meat by direct glycine elution and PEG 8000 precipitation. Total RNA was then extracted from the PEG precipitate by the guanidine thiocyanate method and used in RT-PCR. Enterovirus genomes were detected in 10% of wild shellfish samples, whilst none was present in the aquaculture samples. Since organisms harvested in both growing areas are used for human consumption, the enterovirus contamination observed in this study may highlight a potential public health risk and illustrate the importance of including routine virological analysis of shellfish in Morocco.