Search

The detection of foodborne viruses in bivalve molluscs is a challenging procedure in relation to low virus concentration and to the presence of significant RT-PCR inhibitors. The aim of this study was the development of an efficient direct extraction method for foodborne viral RNA from bivalve molluscs. Using Mengovirus as a surrogate for foodborne viruses, five extraction methods based on RNA release by Trizol were compared on clams and oysters. A procedure consisting of Trizol, PureLink RNA Mini Kit, followed by Cetyltrimethylammonium bromide (CTAB) treatment and LiCl precipitation was found to provide RNA with the highest extraction efficiency and negligible inhibitory effect on real-time RT-PCR. This procedure was further compared to standard extraction method (ISO 15216) using clam, mussel and oyster samples spiked with Hepatitis A virus, Norovirus (NoV) GI and GII as well as bivalve samples naturally contaminated with NoV GI or GII. Results clearly demonstrated that the developed method provided, on average, a recovery 4·3 times higher than the standard reference protocol as well as good repeatability., Significance and Impact of the Study: A direct extraction procedure was developed to recover viral RNA from shellfish with improved efficiency in comparison to reference extraction method (ISO 15216). Without the need for specific equipment, this procedure offers an alternative for performing food safety controls and for risk assessment studies. Given the inclusion in this extraction method of several steps for the efficient removal of food components inhibiting PCR reaction, this approach could serve as a general scheme for the extraction of nucleic acids of other enteric viruses and/or from other food categories., (© 2018 The Society for Applied Microbiology.)

Aim: To improve the efficacy of intercalating dyes to distinguishing between infectious and inactivated hepatitis A virus (HAV) in food., Methods and Results: Different intercalating dyes were evaluated for the discrimination between infectious and thermally inactivated HAV suspensions combining with the RT-qPCR proposed in the ISO 15216. Among them, PMAxx was the best dye in removing the RT-qPCR signal from inactivated HAV. Applied to lettuce and spinach, PMAxx-Triton pretreatment resulted in complete removal of the RT-qPCR signal from inactivated HAV. Likewise, this study demonstrates that this pretreatment is suitable for the discrimination of inactivated HAV in shellfish without further sample dilution. In mussels and oysters, the developed viability RT-qPCR method reduced the signal of inactivated HAV between 1·7 and 2·2 logs at high inoculation level, and signal was completely removed at low inoculation level., Conclusions: This study showed that the use of PMAxx is an important improvement to assess HAV infectivity by RT-qPCR. It was shown that PMAxx-Triton pretreatment is suitable for the analysis of infectious HAV in complex food samples such as vegetables and shellfish., Significance and Impact of the Study: The PMAxx-Triton pretreatment can be easily incorporated to the ISO norm for infectious virus detection., (© 2017 The Society for Applied Microbiology.)

Aims: The aim of this study was to identify the origin of faecal pollution impacting the Elorn estuary (Brittany, France) by applying microbial source tracking (MST) markers in both oysters and estuarine waters., Methods and Results: The MST markers used were as follows: (i) human-, ruminant- and pig-associated Bacteroidales markers by real-time PCR and (ii) human genogroup II and animal genogroup I of F-specific RNA bacteriophages (FRNAPH) by culture/genotyping and by direct real-time reverse-transcriptase PCR. The higher occurrence of the human genogroup II of F-specific RNA bacteriophages using a culture/genotyping method, and human-associated Bacteroidales marker by real-time PCR, allowed the identification of human faecal contamination as the predominant source of contamination in oysters (total of 18 oyster batches tested) and waters (total of 24 water samples tested). The importance of using the intravalvular liquids instead of digestive tissues, when applying host-associated Bacteroidales markers in oysters, was also revealed., Conclusions: This study has shown that the application of a MST toolbox of diverse bacterial and viral methods can provide multiple lines of evidence to identify the predominant source of faecal contamination in shellfish from an estuarine environment., Significance and Impact of the Study: Application of this MST toolbox is a useful approach to understand the origin of faecal contamination in shellfish harvesting areas in an estuarine setting., (© 2013 The Society for Applied Microbiology.)

Aims: 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., Methods and Results: Environmental parameters (temperature and salinity) and hydrometric levels of the tributary river were measured. Seventy shellfish samples (35 samples per area) were examined for Escherichia coli and NoV (GI and GII). NoV contamination was found in 51.4% of samples, of which, 2.9% contained only NoV GI, 14.3% only NoV GII, while the majority of the samples (34.3%) contained both genogroups. Most of the positive results (90.0%) were obtained in the period between November 2008 and April 2009., Conclusions: No significant differences were found between the results from the two harvesting areas and the three shellfish species. However, on the basis of the average C(t) values, the recovery rate (from 0.46 to 1.15%) and the distribution of positive results in the samplings, mussels seem to be a suitable indicator species to monitor viral contamination in these areas., Significance and Impact of the Study: The data allow the optimization of monitoring plans to improve the prevention strategies in terms of money and time, by the intensification of controls in the cold season and the use of one species as indicator., (© 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.)

Aims: To survey for hepatitis A virus (HAV) and hepatitis E virus (HEV) contamination in edible bivalve shellfish., Methods and Results: A total of 213 shellfish (52 oysters, 69 cockles and 92 mussels) collected from a culture farm and two retailed markets were investigated for HAV and HEV contamination by reverse transcription-polymerase chain reaction (RT-PCR) assay using HA2-HA1 (capsid region) and HE366-HE363 (ORF2/3 overlapping region) primers, respectively. It was found that 3.8% of the shellfish and 2.9 and 6.5% of the cockle and mussel, respectively, showed positive for HAV detection. Nucleotide sequencing of all the 8 HAV-positive shellfish revealed 97-100% similarity to HAV subgenotype IA. Interestingly, viruses were found more frequently in the gills than in digestive tissue (4.5%vs 0.5%, P = 0.045). All the shellfish were negative for HEV., Conclusion: Significant contamination of HAV in edible bivalve shellfish was observed. Beside digestive tissue, gills are one of the important samples for viral genome detection., Significance and Impact of the Study: HAV-contaminated shellfish can play a role as reservoirs and/or vehicles in faecal-oral transmission in Thailand, and further monitoring of such a contamination is required., (© 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)

Background: We present the investigation of an outbreak of gastroenteritis at a UK restaurant incorporating both epidemiological and microbiological analysis., Methods: Structured postal questionnaires were sent to 30 diners who ate at the restaurant during the outbreak period (5-7 February 2010). Stool specimens collected from staff and diners were submitted for bacterial culture and norovirus testing, and 15 Pacific oysters (Crassostrea gigas) from the batch served during the outbreak period were tested for norovirus., Results: A strong association was observed between illness and oyster consumption (odds ratio undefined, confidence interval: 11.7 to infinity, P = 0.00001). Multiple different sequences of norovirus RNA were present in both stool and oyster specimens, typical of a shellfish origin. Several contemporaneous norovirus outbreaks throughout the UK were linked to oysters, particularly, though not exclusively, those sourced from Carlingford Lough in Ireland (as in this study), which were subsequently withdrawn from distribution., Conclusion: Despite the risk to human health, there is significant uncertainty surrounding the quantitative correlation between oyster norovirus levels and consumer illness. Continued research should help further our understanding of this crucial correlation and identify ways in which viral depuration of oysters can be enhanced.

Aims: To examine the uptake and tissue distribution of norovirus (NoV) and poliovirus (PV) experimentally bioaccumulated in feeding Pacific oysters (Crassostrea gigas)., Methods and Results: Pacific oysters were allowed to bioaccumulated either PV or NoV under tidally synchronized feeding conditions in laboratory tanks. Oysters were then either fixed and paraffin wax embedded prior to localizing virus within tissues by immunohistochemistry (IHC), or they were dissected into digestive tract (stomach, intestine and digestive diverticula), gill and labial palp tissues, and the viral load determined by quantitative RT-PCR. Both PV and NoV immunoreactivities were predominantly found in the lumen and within cells of the digestive tract tissues; however, PV was also found within cells of nondigestive tract tissues, and in the gills and labial palp. Quantitative RT-PCR of tissue extracts corroborate the immunohistochemical data in that the major site for virus localization is the gut, but significant amounts of viral RNA were identified in the gills and labial palp., Conclusions: The human enteric viruses, PV and NoV, are readily bioaccumulated by feeding Pacific oysters and that some of the virus is internalized within cells of both digestive and nondigestive tissues., Significance and Impact of the Study: Oysters that have been virally contaminated even after depuration (cleaning) in uncontaminated seawater could pose a human health risk if consumed.

Aims: To study the distribution of norovirus (NV) in oyster tissues., Methods and Results: Five monoclonal antibodies (MAbs) against VP1 were selected from Balb/c mice. Immunohistochemical analysis was performed to detect native NV in different tissues of artificially contaminated oysters using the MAbs. The data showed that the gills and the digestive glands are efficient tissues for accumulation of the NV. In addition, the NV was found on the cilia of the mantle after filtering. In our study, only NV RNA in the gills could be detected by reverse transcriptase-polymerase chain reaction., Conclusions: NV was bioaccumulated in the gills, stomach, digestive diverticula and cilia of the mantle. Furthermore, the results suggested that the viral load of the gills and the digestive glands is heavier than that of the other tissues., Significance and Impact of the Study: This, to our knowledge, is the first paper to report the distribution of NV in oyster tissues by immunoassay after artificial contamination. Further understanding of the NV distribution in oyster may help us to sample appropriate tissues for detection of the virus.

Aim: To evaluate the different tissues of naturally contaminated oyster for food-borne virus detection., Methods and Results: The different tissues of 136 field oyster samples were analysed for norovirus (NV), hepatitis A virus (HAV) and rotavirus (RV) by reverse transcription (RT)-PCR and were confirmed by sequencing. These viruses were detected in 20 samples (14.71%), showing positivity for NV (1.47%), HAV (5.15%) and RV (8.82%). Furthermore, among different tissues, the highest positive rate of the food-borne viruses was found in the gills (14.71%), followed by the stomach (13.97%) and the digestive diverticula (13.24%)., Conclusions: The food-borne viruses were detected in the gills, stomach, digestive diverticula and the cilia of the mantle. In addition, the results showed that the gills are one of the appropriate tissues for viral detection in oysters by nucleic acid assay., Significance and Impact of the Study: This is the first paper to report on the presence of food-borne viruses in the gills and the cilia of the mantle of naturally contaminated oysters. The research team hopes that the results of the study will be of help in sampling the appropriate tissues for the detection of food-borne viruses in commercial oysters.

Background: Between November 2003 and January 2004, outbreaks of norovirus in 3 Australian jurisdictions involving 83 cases of illness were associated with imported oyster meat., Methods: Cohort studies were conducted in 2 jurisdictions to identify relative risks of illness for the consumption of oysters. A case series was conducted in the third jurisdiction., Results: The cohort studies conducted in the first 2 jurisdictions identified relative risks of illness of 17 (95% confidence interval, 5-51) and 35 (95% confidence interval, 5-243), respectively, for the consumption of oysters. Multiple strains of norovirus were detected in fecal specimens from 8 of 14 patients and in 1 of the 3 batches of implicated oyster meat using seminested reverse-transcriptase polymerase chain reaction methods. Traceback investigations revealed that all oyster meat was harvested from the same estuary system in Japan within the same month., Conclusions: These outbreaks demonstrate the potential of foodborne disease to spread internationally and the need for national and international collaboration to investigate such outbreaks. Foodborne illness related to norovirus is underestimated because of underreporting of human cases and challenges in laboratory detection of viruses in foods, both of which can delay public health action.

The 39 oyster consumption-related cases of hepatitis A reported in 2005 represent the first large outbreak of hepatitis A associated with shellfish consumption in the United States in >15 years. This is the first outbreak investigation in which an identical hepatitis A virus sequence was obtained from both the implicated food product and case patients.

Aims: To determine if histo-blood group antigens (HBGA) present in oyster gastrointestinal (GI) cells mediate accumulation of human noroviruses (NoV) in oyster GI cells., Methods and Results: HBGA-specific monoclonal antibodies (MAbs) were used to determine the presence of the corresponding HBGA in oyster GI cells. All oyster samples tested contained type A-like HBGA in GI tissue as measured by ELISA. Recombinant Norwalk virus viral like particles (rNVLP) were bound to plates coated with oyster GI homogenate. The binding was inhibited when rNVLPs were pre-incubated with MAbs specific for type A HBGA, or samples of human saliva from type A individuals. Co-localization of rNVLP and type A-like HBGA, but not type B-like or type H-like HBGA, on GI epithelial cells was observed by immunofluorescent histochemical staining and three-channel confocal scanning laser microscopy., Conclusion: Type A-like HBGA is present in oyster GI cells and responsible for binding of rNVLP., Significance and Impact of the Study: This is the first report of the presence of type A-like HBGA in oyster GI cells and the specific binding of rNVLP to type A-like HBGA on oyster GI cells. The results of this study suggest that human NoV concentrate in oyster GI cells by specific binding to concentrated type A-like HBGA rather than by a nonmolecular entrapment within the tissues.

A one-step procedure was developed to confirm viral targets by using a fluorometric 96-well microplate scanner following polymerase chain reaction (PCR). The fluorogenic PCR, integrated with fluorometric scanning, measured the end point fluorescence of viral PCR amplicon/probe hybrids and permitted the use of nonfluorogenic PCR conditions with addition of a Cy3 fluorophore-labeled linear probe for viruses. This linear probe generated higher ratios of viral signal-to-noise than a comparative beacon probe. Detection efficiency with a Cy3/quencher linear probe was comparable with Southern analysis at the level > or = 0.27 plaque-forming units (PFU) of poliovirus/PCR. For the reaction containing < 0.27 PFU, the fluorometric measurements of the first-round PCR viral amplicon were not as sensitive as Southern analysis; however, equivalent sensitivities were achieved with fluorogenic nested PCR. Concentrates of 11 oyster samples exposed to municipal sewage were tested for enteroviruses; the fluorogenic detection correlated 100% with Southern analysis. This method using fluorometric scanning of viral amplicon is simple; it requires neither continuously monitoring equipment nor redesigning PCR primers; and it accurately detects enteroviruses in oyster sample concentrates in less time than classic spectrophotometry or Southern analysis.

Aims: Optimal detection of enteric RNA viruses in clinical, environmental, and food products using reverse transcription-PCR (RT-PCR) when inhibitory substances in extracted sample materials are present., Methods and Results: We adapted a device for detection of RNA viruses in plant tissues and insects to detect a calicivirus strain (San Miguel sea lion virus, serotype 17) in water and oyster tissue extracts. This single, compartmentalized tube-within-a-tube (TWT) device for RT-PCR-nested PCR was compared to a conventional protocol of RT-PCR-nested PCR. In the presence of 100 mg of shellfish tissue extract equivalent, this TWT device decreases the calicivirus assay detection limit 10-fold over that of conventional RT-PCR-nested PCR while maintaining an identical detection limit of viral nucleic acid suspended in water. Both the conventional and TWT methods estimated the total particle-to-infectious particle ratio for this strain of calicivirus at approximately 40 : 1., Conclusions: We believe that the TWT device with appropriate RT-PCR primers will decrease the detection limit for other calicivirus strains and RNA viruses in shellfish tissue extracts., Significance and Impact of the Study: We believe that the TWT approach is applicable to other situations where RT and/or PCR inhibitory materials are present or nucleic acid targets of bacteria or viruses are at low levels in extracts of food products or clinical specimens.

Norwalk-like viruses (NLVs), or small round structured viruses, are known to cause acute gastroenteritis associated with eating contaminated shellfish. Between 1993 and 1996, three oyster-related gastroenteritis outbreaks attributed to NLV occurred in Louisiana. Intensive trace-back and environmental investigations revealed that the overboard disposal of sewage by oyster harvesters into oyster-bed waters was the most likely source of contamination in at least two of the outbreaks. The small infectious dose of NLV, the large quantity of virus particles in stool, and the ability of oysters to concentrate virus particles suggest that oyster-related outbreaks will continue unless strong control measures are established. Efforts to halt improper sewage disposal in oyster-harvesting waters, including overboard sewage discharge, must be undertaken if future outbreaks are to be prevented.

In the 1990s, Norwalk-like viruses (NLVs) were identified in patient specimens as the primary pathogen associated with shellfish-borne gastroenteritis in the United States. Identification of these viruses from implicated shellfish has been difficult due to inefficient recovery of viruses, natural polymerase chain reaction (PCR) inhibitors in shellfish, and low virus contamination. Recent improvements to the method of detecting NLVs in shellfish include enhanced processing of virus and shellfish samples, application of nested PCR and nucleotide sequencing, and increased knowledge of NLV genetic diversity. Using a newly developed and sensitive method, an NLV G2 strain was identified in 2 oyster samples implicated in a 1998 California outbreak involving 171 cases. NLV capsid primers demonstrated a greater specificity of PCR detection than did polymerase primers. The 175-base viral capsid nucleotide sequences derived from oysters were 100% identical to those derived from a patient stool sample. This finding supports the epidemiologic associations indicating that contaminated shellfish serve as the vehicle for NLV transmission.

In order to detect the low numbers of hepatitis A viral (HAV) particles which may potentially be present in food and cause a serious illness, an original procedure which combines immunomagnetic separation and PCR is described. The use of streptavidin magnetic beads coated with biotinylated human anti-HAV IgG allows virus capture and the removal of the RT-PCR inhibitory compounds which usually are present in shellfish extracts. Following immunomagnetic capture, the separated HAV were lysed, the beads discarded, and the supernatant containing the viral RNA subjected to the RT-PCR protocol. Levels of HAV ranging from 10 to 10(5) pfu were successfully detected in artificially contaminated samples of shucked American oyster (Crassostrea virginica).