Three-year study to assess human enteric viruses in shellfish

The main pathogenic enteric viruses able to persist in the environment, such as hepatitis A virus (HAV), Norwalk-like virus (NLV), enterovirus (EV), rotavirus (RV), and astrovirus (AV), were detected by reverse transcription-PCR and hybridization in shellfish during a 3-year study. Oyster samples (n = 108), occasionally containing bacteria, were less frequently contaminated, showing positivity for AV (17%), NLV (23%), EV (19%), and RV (27%), whereas mussel samples, collected in areas routinely impacted by human sewage, were more highly contaminated: AV (50%), HAV (13%), NLV (35%), EV (45%), and RV (52%). Sequences obtained from HAV and NLV amplicons shelved a great variety of strains, especially for NLV (strains close to Mexico, Snow Mountain Agent, or Norwalk virus), Viral contamination was mainly observed during winter months, although there were some seasonal differences among the viruses. This first study of virus detection over a fairly long period of time suggests that routine analysis of shellfish by a molecular technique is feasible

Possible relation between a winter epidemic of acute gastroenteritis in France and viral contamination of shellfish

Several outbreaks of gastroenteritis related to the consumption of shellfish (frequently eaten raw) have been reported in different parts of the world. In Europe, hum an calicivirus infections may have been involved in winter outbreaks in recent years, although there is little evidence confirming such viral contamination in shellfish. This study presents the first results of a field survey on viral contamination in two shelllish harvesting areas along the French Mediterranean coast. The first, consisting mainly of oyster beds, was c1assified in category A, as determined by fecal coliform counts in shellfish (European Community Directive 91/492), and the second, a mussel bed, was c1assified in category C. Shelllish samples were collected monthly between August, 1995 and April, 1997, and RT-PCR was used to detect viruses known to be involved in outbreaks of gastroenteritis: enterovirus, human calicivirus, rotavirus, and astrovirus. Contamination by fecal coliforms was evaluated in the same samples. Virological results in shellfish were correlated with data on the incidence of epidemics of gastroenteritis in the coastal population obtained from a French survey. A relationship was observed between virological results and epidemiological data. For the 2 years when the incidence rate of gastroenteritis was maximal in win ter, the mussel bed \Vas always contanunated by the four types of viruses screened. Sinular results were observed for oyster beds during the second winter; whereas, two sampi es were lughly contarninated during the first winter, and a third showed low contamination (only rotavirus). These results suggest that an epidemic of gastroenteritis in the hum an population contributed ta viral contamination of the marine environment through discharge of waste water

Microalgal sensitivity varies between a diuron-resistant strain and two wild strains when exposed to diuron and irgarol, alone and in mixtures

A wild strain of Chaetoceros calcitrans and wild and diuron-resistant strains of Tetraselmis suecica, were exposed to the PSII inhibitor herbicides diuron and irgarol, individually and in mixtures. The effects of three concentrations of diuron and irgarol and four binary mixtures were evaluated on doubling time, relative reactive oxygen species and lipid content by flow cytometry, and on photosynthetic efficiency by pulse amplitude modulated fluorescence. In both wild strains, significant effects were observed for each molecule at the highest concentration tested: at irgarol 0.5 μg L−1, C. calcitrans was shown to be more sensitive than T. suecica (+52% and +19% in doubling time, respectively), whereas at diuron 5 μg L−1, T. suecica was more affected (+125% in doubling time) than C. calcitrans (+21%). Overall, irgarol had a higher toxicity at a lower concentration than diuron (no effect at diuron 0.5 μg L−1) for both wild strains. The strongest mixture (irgarol 0.5 μg L−1 + diuron 5 μg L−1) increased doubling time by 356% for T. suecica, thus showing amplified effects when the two compounds were mixed. Sequencing of the diuron-resistant strain demonstrated a single mutation in the psbA gene coding sequence. Although resistance of this strain to diuron was confirmed with no effect at the highest diuron concentration, no resistance to irgarol was shown. In addition, the mutant strain exposed to the strongest mixture showed a 3.5-fold increase in doubling time compared with irgarol alone, thereby supporting the hypothesis of a biochemical interaction between these two compounds

Molecular epidemiological survey of rotaviruses in sewage by reverse transcriptase seminested PCR and restriction fragment length polymorphism assay

Rotavirus double-stranded RNA was detected directly in sewage treatment plant samples over a 1-year period by reverse transcription followed by PCR amplification of the VP7 gene and Southern blot hybridization. The presence of naturally occurring rotaviruses was demonstrated in 42% of raw sewage samples and in 67% of treated effluent samples, Amplified viral sequences were analyzed bg restriction enzymes. Ten different restriction profiles were characterized, most of which were found in treated effluent samples. A mixture of restriction profiles was observed in 75% of contaminated effluent samples, The profiles were compared with those obtained from human rotavirus isolates involved in infections in children from the same area (six different profiles were detected), Five identical viral sequences were detected in both environmental and clinical samples, Restriction profiles sere also compared io profiles from known genomic sequences of human and animal viruses. Both human and animal origins of rotavirus contamination of water seemed likely

Validity of Escherichia coli, enterovirus, and F-specific RNA bacteriophages as indicators of viral shellfish contamination

The sanitary classification of harvesting areas for bivalve mollusks in France is based on the level of Escherichia Coli contamination detected in shellfish meat, as defined in EC Directive 91/492 EEC. However, outbreaks of gastroenteritis or hepatitis after consumption of shellfish meeting current bacteriological standards suggest that E. coli is a poor indicator of viral contamination. The purpose of this study was to assess the adequacy of enterovirus and F-specific RNA bacteriophages as new indicators of human enteric viruses. Shellfish were sampled over a 37-mo period to characterize microbial contamination in two coastal areas subjected to different sewage contamination inputs. Contamination by E. coli, F-specific RNA bacteriophages (F+ RNA) and human enteric viruses (enterovirus, EV; hepatitis A virus, HAV; Norwalk-like virus, NLV: astrovirus, AV; and rotavirus, RV) was measured in the same samples. E. coli analysis was performed by conductance measurement, enteric viruses were detected by reverse-transcription polymerase chain reaction (RT-PCR) and hybridization. and F+ RNA was evaluated by culture according to the ISO 10705-1 method. Statistical analysis based on bootstrap methods was performed on 95 series of paired observations. The validity of E. coli, enterovirus, and F-specific RNA bacteriophages as viral indicators was evaluated by measuring their sensitivity and specificity in the presence of enteric viruses. None of the tested indicators proved adequate to protect the public from viral shellfish contamination. The sensitivity of all indicators was better in the highly contaminated zone, and enteroviruses showed the highest specificity for both sites

Bovine Norovirus: Carbohydrate Ligand, Environmental Contamination, and Potential Cross-Species Transmission via Oysters ▿ †

Noroviruses (NoV) are major agents of acute gastroenteritis in humans and the primary pathogens of shellfish-related outbreaks. Previous studies showed that some human strains bind to oyster tissues through carbohydrate ligands that are similar to their human receptors. Thus, based on presentation of shared norovirus carbohydrate ligands, oysters could selectively concentrate animal strains with increased ability to overcome species barriers. In comparison with human GI and GII strains, bovine GIII NoV strains, although frequently detected in bovine feces and waters of two estuaries of Brittany, were seldom detected in oysters grown in these estuaries. Characterization of the carbohydrate ligand from a new GIII strain indicated recognition of the alpha-galactosidase (α-Gal) epitope not expressed by humans, similar to the GIII.2 Newbury2 strain. This ligand was not detectable on oyster tissues, suggesting that oysters may not be able to accumulate substantial amounts of GIII strains due to the lack of shared carbohydrate ligand and that they should be unable to contribute to select GIII strains with an increased ability to recognize humans

Round-robin comparison of methods for the detection of human enteric viruses in lettuce

Five methods that detect human enteric virus contamination in lettuce were compared. To mimic multiple contaminations as observed after sewage contamination, artificial contamination was with human calicivirus and poliovirus and animal calici-virus strains at different concentrations. Nucleic acid extractions were done at the same time in the same laboratory to reduce assay-to-assay variability. Results showed that the two critical steps are the washing step and removal of inhibitors. The more reliable methods (sensitivity, simplicity, low cost) included an elution/concentration step and a commercial kit. Such development of sensitive methods for viral detection in foods other than shellfish is important to improve food safety