Quantitative approach of risk management strategies for hepatitis a virus-contaminated oyster production areas.

It is not yet known whether using the new molecular tools to monitor hepatitis A virus (HAV) in shellfish production areas could be useful for improving food safety. HAV contamination can be acute in coastal areas, such as Brittany, France, where outbreaks of hepatitis A have already occurred and have been linked to the consumption of raw shellfish. A quantitative probabilistic approach was carried out to estimate the mean annual risk of hepatitis A in an adult population of raw oyster consumers. Two hypothetical scenarios of contamination were considered, the first for a rare and brief event and the second for regular and prolonged episodes of contamination. Fourteen monitoring and management strategies were simulated. Their effects were assessed by the relative risk reduction in mean annual risk. The duration of closure after abnormal detection in the shellfish area was also considered. Among the strategies tested, results show that monthly molecular reverse transcription PCR monitoring of HAV is more useful than bacterial surveys. In terms of management measures, early closure of the shellfish area without waiting for confirmatory analysis was shown to be the most efficient strategy. When contamination is very short-lived and homogeneous in the shellfish production area, waiting for three negative results before reopening the area for harvest is time wasting. When contamination is not well identified or if contamination is heterogeneous, it can be harmful not to wait for three negative results. In addition, any preventive measures, such as improving sewage treatment or producing shellfish in safer areas, that can reduce contamination by at least 2 log units are more efficient and less costly. Finally we show that controlling and managing transferred shellfish are useful and can play an important role in preventing cases. Qualitative results from HAV monitoring can advantageously supplement other measures that improve the safety of shellfish products in exposed areas.

A risk-based sampling plan for monitoring of histamine in fish products.

In 2008, the French Institute for Public Health Surveillance reported an increase in the number of histamine food poisoning outbreaks and cases in France. The aim of this study was to propose a new monitoring plan for characterizing consumers' exposure to histamine through fishery products. As fish products of concern are numerous, we proposed that the number of samples allocated for a fish category be chosen based on the risk associated with the category. Point risk estimates of histamine poisoning were assessed with the Risk Ranger tool. Fresh fish with high histidine content was found to contribute most to the number of cases. The (estimated) risks associated with the consumption of canned and deep-frozen fish appear marginal as compared with the risk associated with fresh fish with high histidine concentrations. Accordingly, we recommend excluding canned and deep-frozen fish from the monitoring plan, although these risk estimates can be biased. Within a category, samples were proportional to the relative food consumption of the different fishes. The spatial and seasonal consumption patterns were also taken into account for the design of the new monitoring plan. By testing appropriate numbers of samples from categories of fish products of concern, this plan will permit investigation of trends or comparison of product categories presenting risks of histamine poisoning.

Modeling and evaluation of compliance to water quality regulations in bathing areas on the Daoulas catchment and estuary (France).

The microbiological quality of waters in estuaries determines their acceptability for recreational uses. Microbiological contamination often results from urban wastewater discharges or non-point source pollution (manure spreading), and can cause bathing zones to be closed. European regulations (EC/7/2006) have proposed standards (500 E. coli/100 ml) for the acceptability areas for bathing. In this study, two models were associated to simulate contamination: SWAT on a catchment and MARS 2D in the downstream estuary. After river flow calibration and validation, two scenarios were simulated in SWAT, and E. coli fluxes obtained at the main outlet of the catchment were then introduced into MARS 2D to follow E. coli concentrations in the estuary. An annual evaluation of compliance to bathing area water quality standards was then calculated, linked with daily rainfall classes. Water quality in the estuary was below the standard on 13 days, including 5 days with rainfall superior to 10 mm, due to faecal contamination from soil leaching by rain, and 5 days with rainfall ranging from 0.1 to 5 mm/day, due to the high frequency of this level of rainfall. To conclude, this study allowed us to demonstrate the efficiency of models to gain a better understanding on water quality degradation factors.

An oyster-associated hepatitis A outbreak in France in 2007.

Following the notification of nine hepatitis A cases clustered in the Cotes d Armor district in northwestern France, epidemiological, environmental and microbiological investigations were set up in order to identify the source and vehicle of contamination and implement control measures. In total, 111 cases were identified in the outbreak, all of whom lived or had stayed as tourists in the Cotes d Armor district. Of the cases, 87% had eaten raw shellfish, and 81% specifically oysters. Traceback investigations carried out on raw shellfish consumed by the cases showed that the raw shellfish originated from a single shellfish farm. The shellfish were probably contaminated either in the submersible tanks or in a depuration land-based tank where they were stored. The source of contamination was not identified but shellfish could have been tainted by sewage overflows or by wastewater releases from a polluted storm sewer close to the shellfish farm or from on-site sanitation facilities. To prevent future hepatitis A outbreaks due to shellfish consumption from this area, hazards specific to each farm should be analysed. Timely information on sewage overflows should also be part of communities efforts regarding sewage collection and treatment.

Microbial impact of small tributaries on water and shellfish quality in shallow coastal areas.

This study aimed to investigate the impact of small tributaries on seawater and shellfish quality in coastal area subjected to brief episodes leading to fecal contamination. Escherichia coli and F-RNA-specific bacteriophages were selected as fecal indicators and astroviruses were chosen as being representative of pathogens in the human population during winter viral epidemics. A two-dimensional hydrodynamic model was built to simulate the current and dispersion in the model domain, which includes areas uncovered at low tide. The model also includes decay rates to simulate microorganism behavior and assess the influence of fecal input on shellfish quality. The originality lies in the fact that specific features of the study area were considered. Modeling results indicate limited particle movements and long flushing times at the back of the bay, where shellfish are farmed. Computational results showed that under normal conditions, i.e. 94% of the time, when rainfall was less than 10 mm per day, the sector shows acceptable water quality. These results are in agreement with shellfish concentration measured in the field. Under high flow conditions, high concentrations of fecal indicators and astrovirus were measured in the river and tributaries. The corresponding fluxes were over 50 times higher than under normal weather conditions. The location of the shellfish beds near the coast makes them vulnerable and fecal indicators and viruses were detected in shellfish after short rainfall events. Our modeling approach makes a contribution to shellfish management and consumer protection, by indicating the "risk period" as defined by EU regulations. Molecular development such as viral quantification in conjunction with model developments will help to prevent shellfish contamination and thus provide safer products to consumers and an effective tool for shellfish producers.

First isolation of Shiga toxin 1d producing Escherichia coli variant strains in shellfish from coastal areas in France.

AIMS: This study was carried out to evaluate the presence of Shiga toxin-producing Escherichia coli (STEC) and E. coli O157:H7 in shellfish from French coastal environments. METHODS AND RESULTS: Shellfish were collected in six growing areas or natural beds (B category) and nonfarming areas (D category) from July 2002 to August 2004. PCR detection of stx genes was performed on homogenized whole shellfish and digestive gland tissues enrichments. STEC strains were detected by colony DNA hybridization using a stx-specific gene probe and E. coli O157 strains were additionally searched by immunomagnetic separation with O157-specific magnetic beads. Stx genes were detected in 40 of 144 (27.8%) sample enrichments from mussels, oysters or cockles, 32 of 130 enrichments (24.6%) were from B-category areas and eight of 14 (57.1%) from the D-category area. Five strains carrying stx(1) or stx(1d) genes and one stx negative, eae and ehxA positive E. coli O157:H7 were isolated from six of 40 stx-positive enrichments. No relation was found between the total E. coli counts in shellfish and the presence of STEC strains in the samples. CONCLUSIONS: The STEC strains of different serotypes and stx types are present in shellfish from French coastal environments. It is the first isolation of STEC stx1d strains in France. SIGNIFICANCE AND IMPACT OF THE STUDY: Shellfish collected in coastal environments can serve as a vehicle for STEC transmission.

Molecular epidemiology of caliciviruses detected in sporadic and outbreak cases of gastroenteritis in France from December 1998 to February 2004.

We compiled sequence and epidemiological data from 172 caliciviruses detected in France from December 1998 to February 2004 in sporadic and outbreak cases. The results showed a cocirculation of strains with a majority of genogroup II (GII) noroviruses. Three groups of noroviruses, not detected before in our laboratory, emerged and spread during the period: the recombinant GGIIb and Norwalk-related strains not amplified in the polymerase gene in 2000 and a new Lordsdale variant in 2002. We observed that (i) GII-4 noroviruses were predominant in nursing home and hospital outbreaks but rare in oyster- and water-related outbreaks despite continuous circulation in the population; (ii) at the opposite, genogroup I strains were detected in the majority of environmental outbreaks; (iii) several strains were frequently found in oyster- and water-linked outbreaks (up to seven), whereas one single strain was detected when transmission was from person to person; and (iv) whereas GII noroviruses were predominant in sporadic cases where patients were under 15 years of age, GI strains were more frequent in outbreaks occurring in this age group. Finally, from a methodology point of view, this compilation shows that detection and characterization in the polymerase gene are not adequate in a significant number of cases and should be completed by amplification and sequencing in the capsid gene.

mRNA detection by reverse transcription-PCR for monitoring viability and potential virulence in a pathogenic strain of Vibrio parahaemolyticus in viable but nonculturable state.

AIMS: This work investigates the maintenance of viability and potential virulence of Vibrio parahaemolyticus in a viable but nonculturable population (VBNC) state by reverse transcription-polymerase chain reaction (RT-PCR). METHODS AND RESULTS: Housekeeping genes, 16S-23S rDNA and rpoS, as well as virulence genes, tdh1 and tdh2, were selected and detected by PCR in a pathogenic strain of V. parahaemolyticus (Vp4). Their expression was then studied by RT-PCR in V. parahaemolyticus Vp4 cultivated in rich medium at 37 degrees C. The 16S-23S rDNA and rpoS, tdh1, tdh2 genes were transcripted at the mid-logarithmic, stationary and late stationary phases, corresponding to various physiological states. The expression of these genes was also studied by RT-PCR in a VBNC population of V. parahaemolyticus Vp4 in artificial seawater (ASW). The effect of temperature (washing of bacterial culture and microcosms) on the attaining VBNC bacteria was first considered. Washing of V. parahaemolyticus Vp4, collected at the mid-logarithmic phase, at 10 or 4 degrees C before inoculation in ASW at 4 degrees C allowed bacteria entered the VBNC state between 22 and 31 days. The 16S-23S rDNA and rpoS gene were expressed in the VBNC bacteria whereas no expression of the tdh1 and tdh2 genes was observed in the same populations. CONCLUSION: The two selected housekeeping genes, 16S-23S rDNA and rpoS, proved to be good viability markers for V. parahaemolyticus Vp4 in culturable and VBNC states. These first data indicated that the pathogenic strain Vp4 would not maintain the expression of the virulence genes, tdh1 and tdh2, in VBNC state. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of RT-PCR for investigating the maintenance or not of viability and potential virulence in VBNC V. parahaemolyticus will facilitate further study to evaluate the potential risk presented by this pathogen in the environment.

Real-time RT-PCR for norovirus screening in shellfish.

Real-time RT-PCR, combining amplification and detection of virus-specific amplicons, is a promising tool for norovirus detection in environmental or food samples such as shellfish. We developed a real-time RT-PCR assay based on one-step detection using single primer sets and probes for norovirus genogroups I and II. Seventy and seven RT-PCR units of genogroup I and II reference norovirus strains, respectively, were detected in artificially contaminated oysters. Validation of the new method on 150 archived naturally contaminated shellfish confirmed the utility of the genogroup II primer set to detect a large range of different strains circulating in France since 1995, but genogroup I strains were detected infrequently.

Sewage impact on shellfish microbial contamination.

Coastal areas are frequently contaminated by microorganisms of human origin, due to high population density and low seawater renewal. To evaluate the impact of wastewater input on shellfish quality, a study was conducted in Brittany (France) over a period of 20 months. A hydrodynamic model was used to simulate wastewater impact on microbial water quality. To validate the model, wastewater from the three main sewage treatment plants and shellfish from three sites were sampled monthly. Bacterial indicators (E. coli), F-RNA phages were searched for by culture and noroviruses by RT-PCR and hybridisation. These microorganisms were detected in the three effluents and clams, with no marked seasonal variation. The microbial concentrations in the two oyster beds, distant from the effluent outfall, were low, and only three of the samples were positive for norovirus. For simulation, the winter wastewater inputs of E. coli and phages were calculated and an estimation for norovirus flux was made from the epidemic situation in the population. The microbial behaviour was included in the model by a decay-rate factor. Results from the model calculations were found to be very similar to E. coli and phage concentrations observed in shellfish. For noroviruses, the model indicated that shellfish distant from the wastewater input were under the detection limit of the RT-PCR method. This study demonstrated the use of modelisation to interpret norovirus contamination in various areas.

Calibration of the impedance method for rapid quantitative estimation of Escherichia coli in live marine bivalve molluscs.

AIM: Calibration of impedance measurement was performed vs the Association Françoise de Normalisation (AFNOR) MPN method with a view to rapid enumeration of Escherichia coli in live marine bivalve molluscs. METHODS AND RESULTS: Linear regression models between log10 MPN and detection time (DT) were adjusted for several shellfish types, growth media, and impedance instruments (BacTrac and Malthus systems). Escherichia coli concentrations could be estimated from DT using a single regression line for BacTrac 4100 with M1 medium (R2 = 87.8%) and Malthus with M2 medium (R2 = 86.7%), and two regression lines for BacTrac 4110 with M2 medium (R2 = 86.4 and 88.2%). The uncertainty of the predicted bacterial concentration was around +/-0.43 log unit for duplicate sample analysis. The impedance signal was attributable to E. coli in 99% of cases. All cultures containing E. coli produced an impedance signal with BacTrac 4100 and BacTrac 4110, whereas 5.6% did not exhibit a signal with Malthus. CONCLUSIONS: Impedance measurement is a possible alternative to the MPN method for rapid quantitative estimation of E. coli in live bivalve shellfish. SIGNIFICANCE AND IMPACT OF THE STUDY: The impedance method reduces analysis handling time considerably and is much easier to use than the MPN method. Moreover, results can be obtained within 5-10 h, allowing rapid intervention to ensure public health protection in case of shellfish contamination.

Occurrence of pathogenic vibrios in coastal areas of France.

AIMS: This study was carried out to investigate the occurrence of potentially pathogenic species of Vibrio in French marine and estuarine environments. METHODS AND RESULTS: Samples of coastal waters and mussels collected between July and September 1999 were analysed by culture, using selective media including thiosulphate-citrate-bile salts-sucrose and modified cellobiose-polymixin B-colistin agar. Presumptive Vibrio colonies were isolated and identified using selected biochemical tests. Specific primers based on flanking sequences of the cytolysin, vvhA gene, pR72H DNA fragment and 16S-23S rRNA intergenic spacer region (ISR) were used in a polymerase chain reaction (PCR) to confirm the identification of Vibrio vulnificus, V. parahaemolyticus and V. cholerae, respectively. In this study, V. alginolyticus (99 of 189) was the predominant species, followed by V. parahaemolyticus (41 of 189), V. vulnificus (20 of 189) and non-O1/non-O139 V. cholerae (three of 189). All 20 V. vulnificus isolates showed PCR amplification of the vvhA gene, 16 of which had been isolated from estuarine water. The PCR amplification of the pR72H DNA fragment in 41 V. parahaemolyticus isolates generated two unique amplicons of 387 and 320 bp. The latter, present in 24.4% of these isolates, had not previously been found in V. parahaemolyticus strains examined to date. Amplification of the trh gene in two of the isolates suggested these to be virulent strains. Three strains identified as V. cholerae by amplification of the 16S-23S rRNA ISR were confirmed to be non-cholera (non-O1/non-O139) strains. CONCLUSIONS: The results of this study demonstrated the presence of pathogenic Vibrio species in French coastal waters. Furthermore, the PCR approach proved useful for the rapid and reliable confirmation of species identification. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate the potential sanitary risk associated with the presence of pathogenic Vibrio spp. in cultivated mussels and in the aquatic environment. The PCR can be used to detect pathogenic vibrios directly in environmental samples.

Astrovirus detection in wastewater samples.

Procedures for the detection of astroviruses in wastewater samples have been developed and evaluated. Following these methodologies, we investigated the occurrence of astroviruses in wastewater samples from three different sewage treatments plants located in Southern France and two in the Barcelona area. Some positive samples were genotyped by analysis of a fragment of the ORF1a by restriction fragment length polymorphism (RFLP) with endonuclease DdeI. The amplimers generated contain several sites for the DdeI restriction endonuclease, being the number and location of sites different between strains.

An improved method for the detection of Norwalk-like caliciviruses in environmental samples.

An original magnetic beads RNA capture was developed for the detection of Norwalk-like virus by RT-PCR. The same oligonucleotide was used both for capture and reverse transcription of the viral RNA. The optimization studies showed that the most important parameter for sensitivity is the biotin-binding capacity of the beads. This method was found to be efficient for eliminating inhibitors in sewage samples compared with the classic RT-PCR. Moreover, the sensitivity was greatly enhanced, allowing the detection of 42% positive sample after gel electrophoresis, which is fourfold greater than classic RT-PCR (11%). Beads-RT-PCR sensitivity is the same as classic RT-PCR and hybridization. Thus, this method, which is easy to perform, should be of particular interest for developing quantitative RT-PCR and sequencing.

[Magnitude of rainfall on viral contamination of the marine environment during gastroenteritis epidemics in human coastal population]. / Importance de la pluviométrie sur la contamination virale du milieu littoral lors de phénomènes épidémiques dans la population.

BACKGROUND: Sewage treatments are not efficient to eliminate enteric microorganisms. Viruses are able to persist and are discharged into the marine environment with treated effluents. Few data are now available on the magnitude and the contributive processes of marine viral contamination. This work evaluates the relationship between the magnitude of rainfall and the viral contamination of the marine environment during winter epidemics of gastroenteritis in human coastal populations. METHODS: A RT-PCR method was used to detect enterovirus, hepatitis A virus, Norwalk-like virus, astrovirus and rotavirus in shellfish, harvested monthly between August 1995 and July 1998. The frequency of virus detection in shellfish was expressed as an Index of Viral Contamination. Acute gastroenteritis in the population was estimated using the French Sentinel System for Monitoring of Communicable Diseases. Rainfall effects on the efficiency of sewage treatment were assessed using an estimated staying time of sewage effluents in the plant. RESULTS: The results indicate that the highest viral contamination occurs in winter. Maximal indexes of viral contamination were respectively 70% in January 1996, 100% in January 1997, but only 31% in January 1998. Viral contamination variations seemed to follow the pattern of the winter epidemic of acute gastroenteritis in the local population in 1996 and 1997. These observations should be linked to the winter rainfalls. Heavy rains on short periods of time could create an hydraulic overload in the sewage treatment plant, reducing the staying time of the sewage effluents and thus the efficiency of the disinfection process. CONCLUSION: The magnitude of the viral contamination of shellfish seems to result from the simultaneity between the winter epidemics of acute gastroenteritis in the coastal population and heavy rainfall. To prevent public health hazards associated with shellfish consumption, the monitoring of microbiological quality in shellfish harvesting areas should include accompagning survey of viral epidemic in the coastal population, and of sewage outputs in the coastal environment.

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 showed 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.

Effect of chlorination on beta-D-galactosidase activity of sewage bacteria and Escherichia coli.

The effect of chlorine on beta-D-galactosidase activity of sewage bacteria and Escherichia coli was studied. beta-D-galactosidase activity of sewage was more resistant to chlorine than faecal coliform cultivability. At low initial dosage (0.05 mg Cl2 l-1) neither cultivability (colony-forming units (cfu)), nor enzyme activity of E. coli suspensions were severely impaired. When initial chlorine concentration was increased to 0.1 mg Cl2 l-1, the cfu number decreased whereas enzyme activity remained high, i.e. the enzyme activity calculated cfu-1 increased. At higher chlorine doses both cfu and enzyme activity were reduced, but non-cultivable cells retained assayable activity after chlorination. Mean values of the enzyme activity calculated cfu-1 decreased when the chlorine dosage was increased from 0.1 to 0.5 mg Cl2 l-1, but were not significantly different (P > 0.05) for dosages of 0.2-0.7 mg Cl2 l-1. After chlorination, beta-D-galactosidase activity of E. coli was less reduced than cfu and direct viable count numbers, but more reduced than 5-cyano-2-3, ditolyl tetrazolium chloride and total cell counts, and the enzyme activity represented an alternative activity parameter of chlorinated samples.

Molecular approaches to measuring microbial marine pollution.

Developments in the rapid detection of pathogens (PCR and its variations) and molecular typing of strains isolated from the ecosystem illustrate the stimulation of research due to the recent foodborne and waterborne disease outbreaks.

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 by 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 were also compared to profiles from known genomic sequences of human and animal viruses. Both human and animal origins of rotavirus contamination of water seemed likely.

Survival of Escherichia coli exposed to visible light in seawater: analysis of rpoS-dependent effects.

We investigated the effect of visible light on Escherichia coli in seawater microcosms. Escherichia coli lost its ability to form colonies in marine environments when exposed to artificial continuous visible light. Survival of illuminated bacteria during the stationary phase was drastically reduced in the absence of the sigma factor (RpoS or KatF) that regulates numerous genes induced in this phase. In the stationary phase, double catalase mutants katE katG and mutants defective in the protein Dps (both catalase and Dps are involved in resistance to hydrogen peroxide (H2O2)), were more sensitive to light. In the exponenital phase, a mutation in oxyR, the regulatory gene of the adaptive response to H2O2, increased sensitivity to light, further suggesting that deleterious effects might be associated with H2O2 production. However, in the stationary phase, the katE katG dps mutant was considerably more resistant to visible light than the rpoS mutant, suggesting rpoS-dependent protection against deleterious effects other than those related to H2O2. The deleterious action of visible light was less important when the salinity decreased. In freshwater, rpoS and katE katG dps mutants did not show a drastic difference in sensitivity to light suggesting that osmolarity sensitizes E. coli to those deleterious effects of visible light that are unrelated to H2O2.