Norovirus recognizes histo-blood group antigens on gastrointestinal cells of clams, mussels, and oysters: a possible mechanism of bioaccumulation

Outbreaks of norovirus (NoV) gastroenteritis are often associated with the consumption of contaminated bivalves such as oysters, clams, and mussels. Crassostrea virginica oysters trap the Norwalk virus through the intestinal type A-like histo-blood group antigen (HBGA), a possible mechanism of bioaccumulation responsible for NoV outbreaks. In this study, we tested binding and inhibition of binding in three species of oysters and one species each of clams and mussels with NoVs, representing four HBGA receptor-binding patterns. Our results indicated that all three oyster species expressed type A- and type O-like HBGA in their gastrointestinal tissue. Similar type A-like antigens also were found in mussels and clams, but only some of them express the O-like antigens. Both genogroups I and II recombinant norovirus-like particles (rNoVLPs) bound to gastrointestinal homogenates from oysters, mussels, and clams, and the binding was inhibited by preincubation of the rNoVLP with HBGA-specific monoclonal antibodies or with types A or O HBGA-positive human saliva. Co-localization of rNoVLPs and HBGA on gastrointestinal epithelial cells of oysters, mussels, and clams was also observed by immunofluorescent microscopy. Finally, the binding of rNoVLP to oyster gastrointestinal homogenates was inhibited by incubation with HBGA analogs. This study significantly expands our understanding that multiple HBGAs are expressed in oyster, mussel, and clam gastrointestinal tissues, which could be the major mechanism of bioaccumulation of NoVs by these bivalves. Our results also suggest that this bioaccumulation could be reversed by incubation with HBGA analogs, a possible important new strategy for depuration.     

Seasonal variation in the chemical composition and fatty acid profile of Pacific oysters (Crassostrea gigas)

The chemical composition and fatty acid profile of the tray-cultured Pacific oyster (Crassostrea gigas) were measured and compared over a 13 month period. Oysters (120–150 g) were cultured in Cork Harbour and samples taken for analysis at monthly intervals. The moisture, fat, protein, glycogen and ash contents of the flesh were analysed. The fatty acid profile of the flesh was analysed by gas chromatography. The condition indices of the oysters were determined. The chemical composition of the oysters grown in Cork Harbour was similar to literature values for the same species grown in Pacific waters. Ranges for the chemical composition (dry flesh weight basis) were: fat (7.8–8.7%), protein (39.1–53.1%), glycogen (21.6–38.9%) and ash (4.0–12.1%).     

长牡蛎中类HBGAs的分型及与诺如病毒P粒子 结合特性研究

目的了解长牡蛎(Crassostrea gigas)消化道组织中类组织血型抗原(histo-blood group antigens,HBGAs)的类型特点,分析其与诺如病毒的结合特性,以探讨长牡蛎富集诺如病毒的机制。方法利用8种HBGAs单克隆抗体,建立长牡蛎中类HBGAs检测的ELISA方法,并分析其主要型别。同时,利用5种体外表达的GII.4型诺如病毒P粒子分析其与长牡蛎中类HBGA的结合特性。结果长牡蛎消化道组织中存在类A,H1,Lea和Ley型HBGA;55019株(2006b变异株)和97-1l株(95/96US变异株)GII.4型诺如病毒P粒子可通过类A、H1和Ley型HBGA与长牡蛎消化道组织相结合,91(Camberwell_91株)和42(Hunter_2004)株可通过类Ley型HBGA与长牡蛎消化道组织相结合,156株(sakai株)不与任何类型类HBGA结合。结论长牡蛎消化道组织中存在类A、H1、Lea和Ley型HBGA,GII.4型诺如病毒主要通过类A、H1和Ley型HBGA与长牡蛎消化道组织相结合。     

Norovirus contamination on French marketed oysters

Contaminated shellfish have been implicated in gastroenteritis outbreaks in different countries. As no regulation has been set up yet regarding viral contamination of food, very few data are available on the prevalence of contaminated products on the market. This study presents data obtained from oysters collected on the French market in one producing area over a 16 month period of time. Noroviruses were detected in 9% of samples with a seasonal impact and influence of climatic events. Contamination levels were low and, surprisingly, oysters sampled directly from the producer were found to have less contamination than oysters from supermarkets.     

Surveillance of Enteric Viruses and Microbial Indicators in the Eastern Oysters (Crassostrea virginica) and Harvest Waters along Louisiana Gulf Coast

Noroviruses are the most common causative agent of viral gastroenteritis in humans, and are responsible for major foodborne illnesses in the United States. Filter‐feeding molluscan shellfish exposed to sewage‐contaminated waters bioaccumulate viruses, and if consumed raw, transmit the viruses to humans and cause illness. We investigated the occurrence of norovirus GI and GII and microbial indicators of fecal contamination in the eastern oysters (Crassostrea virginica) and water from commercial harvesting areas along the Louisiana Gulf Coast (January to November of 2013). Microbial indicators (aerobic plate count, enterococci, fecal coliforms, Escherichia coli, male‐specific coliphages, and somatic coliphages) were detected at the densities lower than public health concerns. Only one oyster sample was positive for norovirus GII at 3.5 ± 0.2 log₁₀ genomic equivalent copies/g digestive tissues. A stool specimen obtained from an infected individual associated with a norovirus outbreak and the suspected oysters (Cameron Parish, La., area 30, January 2013) were also analyzed. The norovirus strain in the stool belonged to GII.4 Sydney; however, the oysters were negative and could not be linked. In general, no temporal trend was observed in the microbial indicators. Low correlation among bacterial indicators was observed in oysters. Strongest correlations among microbial indicators were observed between enterococci and fecal coliforms (r = 0.63) and between enterococci and E. coli (r = 0.64) in water (P < 0.05); however, weak correlations were found in oysters (r < 0.45) and between oysters and harvest water (r ≤ 0.36, P > 0.05). Our results emphasize the need for regular monitoring of pathogenic viruses in commercial oyster harvesting areas to reduce the risks of viral gastroenteritis incidences.     

Volatile Compounds Characterizing the Aroma of Fresh Atlantic and Pacific Oysters

Volatile compounds which contribute to the distinct melon-like aroma of fresh Pacific oysters (Crassostrea gigas) were identified as (E,Z)-2,6-nonadienal and 3,6-nonadien-1-ol, but these compounds were not found in the less intensely flavored Atlantic oysters (Crassostrea virginica). 1-Octen-3-one, 1-octen-3-ol, 1,5-octadien-3-one, 1,5-octadien-3-ol, and 2,5-octadien-1-ol were found in both species of oysters, and contributed a characteristic heavy, planty aroma to each. Sensory panel scores for intensity of fresh melon-like flavors were significantly higher for both raw and steamed Pacific oysters than for comparable Atlantic oysters. Enzymic generation of alcohols and carbonyls that comprise the characterizing aroma compounds from polyunsaturated fatty acids was demonstrated by preventing their formation with specific inhibitors for cyclooxygenase (acetylsalicylic acid) and lipoxygenase [tin (II) chloride].     

Reductions of Vibrio parahaemolyticus in Pacific oysters (Crassostrea gigas) by depuration at various temperatures

Consumption of raw oysters has been linked to several outbreaks of Vibrio parahaemolyticus infection in the United States. This study investigated effects of ice storage and UV-sterilized seawater depuration at various temperatures on reducing V. parahaemolyticus in oysters. Raw Pacific oysters (Crassostrea gigas) were inoculated with a mixed culture of five clinical strains of V. parahaemolyticus (10290, 10292, 10293, BE 98-2029 and 027-1c1) at levels of 10⁴⁻⁶ MPN/g. Inoculated oysters were either stored in ice or depurated in recirculating artificial seawater at 2, 3, 7, 10, 12.5, and 15 °C for 4–6 days. Holding oysters in ice or depuration of oysters in recirculating seawater at 2 or 3 °C for 4 days did not result in significant reductions (P > 0.05) of V. parahaemolyticus in the oysters. However, depuration at temperatures between 7 and 15 °C reduced V. parahaemolyticus populations in oysters by >3.0 log MPN/g after 5 days with no loss of oysters. Depuration at refrigerated temperatures (7–15 °C) can be applied as a post-harvest treatment for reducing V. parahaemolyticus in Pacific oysters.     

Rapid virus detection procedure for molecular tracing of shellfish associated with disease outbreaks

Detection of pathogenic viruses in oysters implicated in gastroenteritis outbreaks is often hampered by time-consuming, specialist virus extraction methods. Five virus RNA extraction methods were evaluated with respect to performance characteristics and sensitivity on artificially contaminated oyster digestive glands. The two most promising procedures were further evaluated on bioaccumulated and naturally contaminated oysters. The most efficient method was used to trace the source in an outbreak situation. Out of five RNA extraction protocols, PEG precipitation and the RNeasy Kit performed best with norovirus genogroup III-spiked digestive glands. Analyzing 24-h bioaccumulated oysters revealed a slightly better sensitivity with PEG precipitation, but the RNeasy Kit was less prone to concentrate inhibitors. The latter procedure demonstrated the presence of human noroviruses in naturally contaminated oysters and oysters implicated in an outbreak. In this outbreak, in four out of nine individually analyzed digestive glands, norovirus was detected. In one of the oysters and in one of the fecal samples of the clinical cases, identical norovirus strains were detected. A standard and rapid virus extraction method using the RNeasy Kit appeared to be most useful in tracing shellfish as the source in gastroenteritis outbreaks, and to be able to make effective and timely risk management decisions.     

Effect of High-Pressure Processing on Vibrio parahaemolyticus Strains in Pure Culture and Pacific Oysters

Different strains of Vibrio parahaemolyticus (Vp) in broth cultures and Vp‐inoculated live Pacific oysters (Crassostrea gigas) were subjected to high‐pressure processing (HPP) at 241, 276, 310, and 345 MPa. Results showed Vp numbers were reduced by HPP in both pure culture and whole oysters. Vp inactivation was dependent on time and pressure. Optimum conditions for reducing Vp in pure culture and oysters to nondetectable levels were achieved at 345 MPa for 30 and 90 s, respectively. Resistance variations were detected between Vp in pure culture and in oysters. HPP proved to be an efficient means of reducing Vp in oysters.     

北部湾海区几种常见牡蛎基础营养及脂肪酸成分比较分析

目的 比较分析北部湾海区常见的熊本牡蛎、香港牡蛎和近江牡蛎鲜品的基础营养成分及脂肪酸组成。方法 以北部湾海区熊本牡蛎、香港牡蛎和近江牡蛎为实验样品, 测定其水分、粗蛋白、粗脂肪和灰分的含量, 并用气相色谱-质谱联用法(gas chromatography-mass spectrometry, GC-MS)测定脂肪酸组成及含量。 结果 香港牡蛎的水分含量(89.44 g/100 g)、近江牡蛎粗脂肪含量(1.09 g/100 g)和灰分含量(1.77 g/100 g)均显著高于另两种牡蛎(P<0.05), 香港牡蛎粗蛋白含量(4.59 g/100 g)显著低于另两种牡蛎(P>0.05)。熊本牡蛎和近江牡蛎含有33种脂肪酸, 而香港牡蛎为34种, 其中多不饱和脂肪酸(polyunsaturated fatty acid, PUFA)含量占41.02%~50.79%; 3种牡蛎中二十碳五烯酸(eicosapentaenoic acid, EPA)、二十二碳六烯酸(docosahexoenoic acid, DHA)的含量占脂肪酸总量的27.13%~38.65%, 近江牡蛎(27.13%)显著低于另两种牡蛎(P<0.05)。结论 北部湾海区熊本牡蛎、香港牡蛎和近江牡蛎脂肪酸种类丰富, 与饱和脂肪酸及单不饱和脂肪酸含量所占脂肪酸总量相比, PUFA占比最高, 且以DHA和EPA为主, 具有优良的开发潜力。     

Determination of norovirus contamination in oysters from two commercial harvesting areas over an extended period, using semiquantitative real-time reverse transcription PCR

The human health risk associated with the consumption of molluscan shellfish grown in sewage-contaminated waters is well established. Noroviruses, which cause gastroenteritis, are the principal agents of shellfish-related illness. Fecal-indicator quality standards based on Escherichia coli are well established in Europe and elsewhere. However, norovirus outbreaks after consumption of shellfish meeting these standards still occur, and the need to improve consumer health protection is well recognized. Alternative approaches proposed include direct monitoring of viral pathogens and the use of alternative indicator organisms capable of providing a better indication of virus risk. This study applies a recently developed TaqMan PCR assay to assess norovirus contamination in shellfish. Comparison was made with E. coli as the existing sanitary standard and a male-specific RNA bacteriophage as a possible alternative. Two commercial pacific oyster (Crassostrea gigas) harvesting areas were monitored over a 31-month period. The results show peaks of norovirus contamination in both areas during winter months, with average levels approximately 17 times higher in oysters sampled October to March than during the remainder of the year, consistent with epidemiological data for the United Kingdom showing oyster-associated illness is confined to winter months. While there was no apparent association with E. coli, an association between levels of norovirus contamination and the male-specific RNA bacteriophage was noted, with average norovirus levels over 40 times higher in samples with male-specific RNA bacteriophage counts of >1,000 PFU/100 g than in samples with <100 PFU/100 g. Overall, these results suggest that norovirus monitoring in shellfish production areas could be an effective strategy for reduction of virus risk.     

北部湾海区三种常见牡蛎的蛋白质及氨基酸营养分析与评价

为探究北部湾海区常见的熊本牡蛎、香港牡蛎和近江牡蛎三种牡蛎的蛋白质和氨基酸含量、蛋白质营养价值及基于氨基酸含量的综合品质评价差异.采用国家标准测定牡蛎中蛋白质含量,利用高效液相色谱法测定牡蛎中氨基酸组成及含量,分别进行蛋白质营养价值评价,并以牡蛎中的氨基酸组成和含量为指标进行主成分分析(Principal compon...     

Evaluation of nonisotopic DNA hybridization methods for detection of the tdh gene of vibrio parahaemolyticus

Production of the thermostable direct hemolysin (TDH) by Vibrio parahaemolyticus is associated with pathogenicity of the organism and is encoded by the tdh gene. The timely resolution of seafood-associated outbreaks requires rapid and accurate detection of pathogenic V. parahaemolyticus. The specificity of alkaline phosphatase- and digoxigenin-labeled tdh gene probes was evaluated against 61 strains of V. parahaemolyticus (including isolates from recent outbreaks involving oysters from the Pacific Northwest, Texas, and New York), 85 strains of other vibrios, and 7 strains of non-vibrio species from clinical and environmental sources. The probes were specific for detection of the V. parahaemolyticus tdh gene.     

Effect of intertidal exposure on Vibrio parahaemolyticus levels in Pacific Northwest oysters

Interest in Vibrio parahaemolyticus (Vp) increased in the United States following Vp-associated gastroenteritis outbreaks in 1997 and 1998 involving the West Coast and other areas. The present study evaluated multiple aspects of Vp ecology in the Pacific Northwest with three objectives: (i) to determine the effect of low-tide exposure on Vp levels in oysters, (ii) to determine the relationship between total and pathogenic Vp, and (iii) to examine sediments and aquatic fauna as reservoirs for pathogenic Vp. Samples were collected from intertidal reefs along Hood Canal, Wash., in August 2001. Fecal matter from marine mammals and aquatic birds as well as intestinal contents from bottom-dwelling fish were tested. Total and pathogenic Vp levels in all the samples were enumerated with colony hybridization procedures using DNA probes that targeted the thermolabile direct hemolysin (tlh) and thermostable direct hemolysin (tdh) genes, respectively. The mean Vp densities in oysters were four to eight times greater at maximum exposure than at the corresponding first exposure. While tdh-positive Vp counts were generally < or = 10 CFU/g at first exposure, counts as high as 160 CFU/g were found at maximum exposure. Vp concentrations in sediments were not significantly different from those in oysters at maximum exposure. Pathogenic (tdh positive) Vp was detected in 9 of 42 (21%) oyster samples at maximum exposure, in 5 of 19 (26%) sediment samples, but in 0 of 9 excreta samples. These results demonstrate that summer conditions permit the multiplication of Vp in oysters exposed by a receding tide.     

Populations of Vibrio parahaemolyticus in retail oysters from Florida using two methods

Vibrio parahaemolyticus is a naturally occurring estuarine bacterium that is often associated with gastroenteritis in humans following consumption of raw molluscan shellfish. A number of studies have investigated the environmental distribution of V. parahaemolyticus, but little is known about the levels of this organism during distribution of oysters or at the point of consumption. Duplicate samples of shellstock oysters were collected monthly (September 1997 to May 1998) from the same four restaurants and three wholesale seafood markets in the Gainesville, Fla. area and analyzed for total V. parahaemolyticus densities using two methods: a standard MPN method (BAM-MPN) and a new direct plating procedure (direct-VPAP). Both methods employed an alkaline phosphatase-labeled DNA probe (VPAP) targeting the species-specific thermolabile hemolysin (tlh) gene to confirm suspect colonies as V. parahaemolyticus. The highest monthly geometric mean V. parahaemolyticus density was observed in October of 1997 (approximately 3,000/g) with similarly high values during September and November of 1997. From December 1997 to May 1998 mean densities were generally less than 100/g, falling to approximately 10/g in February and March. A strong correlation (r = 0.78) between the direct-VPAP and BAM-MPN methods for determining V. parahaemolyticus densities in market-level oysters was observed. The direct-VPAP method was more rapid and precise while the BAM-MPN was more sensitive and may better recover stressed cells. The utilization of the VPAP probe for identification of V. parahaemolyticus sharply reduced the labor for either method compared to biochemical identification techniques used in earlier V. parahaemolyticus surveys.     

生鲜食品中诺如病毒污染与作用受体的研究进展

诺如病毒(noroviruses,NoVs)是世界范围内引起非细菌性急性胃肠炎疫情爆发的主要病原体。组织血型抗原(histo-blood group antigens,HBGAs)作为NoVs的作用受体,在NoVs的传播中起着极其重要的作用。果蔬产品在从农场到餐桌这一供应链的任何环节均有可能被NoVs污染,已成为传播NoVs的高风险食品。海产贝类作为No Vs的另一重要传播媒介,主要通过滤食作用富集No Vs。部分生鲜食品内也存在类HBGAs与NoVs特异结合,使存在于生鲜食品内的NoVs难以被净化。本文主要综述了NoVs在生鲜食品中的蓄积、分布、污染来源及其作用受体的研究进展。     

Climate anomalies and the increasing risk of Vibrio parahaemolyticus and Vibrio vulnificus illnesses

We examined the potential influence of climate anomalies in expanding the geographical and seasonal range of seafood-borne illnesses from Vibrio parahaemolyticus and Vibrio vulnificus. Archived climate data from areas of implicated seafood production were obtained from various sources, including in situ monitoring devices and satellite imagery. The geographical expansion of V. parahaemolyticus outbreaks into Peru and Alaska corresponded closely with climate anomalies such as El Niño, which brought large masses of abnormally warm water into these regions. Seasonal expansion of V. vulnificus illnesses associated with oysters harvested from the Gulf of Mexico in April and November correspond with warmer water temperatures (>20 °C) recorded during these months since 1998. This retrospective review indicates that climate anomalies have already greatly expanded the risk area and season for vibrio illnesses and suggest that these events can be forecasted. Certainly, when similar circumstances occur in the future, adjustments in industry practices and regulatory policy should be considered, especially for seafood that is consumed raw, such as bivalve mollusks.     

Depuration and Relaying: A Review on Potential Removal of Norovirus from Oysters

Pollution of coastal waters can result in contamination of bivalve shellfish with human enteric viruses, including norovirus (NoV), and oysters are commonly implicated in outbreaks. Depuration is a postharvest treatment involving placement of shellfish in tanks of clean seawater to reduce contaminant levels; this review focuses on the efficacy of depuration in reducing NoV in oysters. There have been many NoV outbreaks from depurated oysters containing around 10³ genome copies/g oyster tissue, far exceeding the median infectious dose (ID50). Half of the published NoV reduction experiments showed no decrease in NoV during depuration, and in the remaining studies it took between 9 and 45.5 d for a 1‐log reduction—significantly longer than commercial depuration time frames. Surrogate viruses are more rapidly depurated than NoV; the mean number of days to reduce NoV by 1 log is 19, and 7.5 d for surrogates. Thus, surrogates do not appear to be suitable for assessing virological safety of depurated oysters; data on reduction of NoV infectivity during depuration would assist evaluations on surrogate viruses and the impact of methods used. The longer persistence of NoV highlights its special relationship with oysters, which involves the binding of NoV to histo‐blood group‐like ligands in various tissues. Given the persistence of NoV and on‐going outbreaks, depuration as currently performed appears ineffective in guaranteeing virologically safe oysters. Conversely, relaying oysters for 4 wk is more successful, with low NoV concentrations and no illnesses associated with products. The ineffectiveness of depuration emphasizes the need for coastal water quality to be improved to ensure oysters are safe to eat.