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.     

Effects of High‐Hydrostatic Pressure on Inactivation of Human Norovirus and Physical and Sensory Characteristics of Oysters

The purpose of the study was to determine the effect of high‐hydrostatic pressure (HHP) on inactivation of human norovirus (HuNoV) in oysters and to evaluate organoleptic characteristics of oysters treated at pressure levels required for HuNoV inactivation. Genogroup I.1 (GI.1) or Genogroup II.4 (GII.4) HuNoV was inoculated into oysters and treated at 300 to 600 MPa at 25 and 0 °C for 2 min. After HHP, viral particles were extracted by porcine gastric mucin‐conjugated magnetic beads (PGM‐MBs) and viral RNA was quantified by real‐time RT‐PCR. Lower initial temperature (0 °C) significantly enhanced HHP inactivation of HuNoV compared to ambient temperature (25 °C; P < 0.05). HHP at 350 and 500 MPa at 0 °C could achieve more than 4 log₁₀ reduction of GII.4 and GI.1 HuNoV in oysters, respectively. HHP treatments did not significantly change color or texture of oyster tissue. A 1‐ to 5‐scale hedonic sensory evaluation on appearance, aroma, color, and overall acceptability showed that pressure‐treated oysters received significantly higher quality scores than the untreated control (P < 0.05). Elevated pressure levels at 450 and 500 MPa did not significantly affect scores compared to 300 MPa at 0 °C, indicating increasing pressure level did not affect sensory acceptability of oysters. Oysters treated at 0 °C had slightly lower acceptability than the group treated at room temperature on day 1 (P < 0.05), but after 1 wk storage, no significant difference in sensory attributes and consumer desirability was observed (P > 0.05).     

2020~2021年广州市售牡蛎中GII型诺如病毒污染情况调查

滤食性牡蛎是食源性诺如病毒传播的重要食品媒介。为了解广州市售牡蛎中的诺如病毒污染水平与遗传多样性特点,合理评估消费风险,本研究于2020年6月至2021年5月期间,每月从当地水产市场随机采集牡蛎样本,采用实验室前期建立的蛋白酶K处理偶联聚乙二醇沉淀小体系法,包括荧光定量RT-PCR和巢式RT-PCR技术检测贝类中病毒的污染量以及基因型分布特点。结果共检测牡蛎110只,GII型诺如病毒阳性检出率为52.7%（58/110）,病毒污染含量范围为1.56×103~1.09×106 copies/g（消化腺）。其中,春夏季节（3~8月）牡蛎中诺如病毒的阳性率为35.7%（20/56）,低于在秋冬季节（9~2月）的阳性率70.4%（38/54）;但不同季节中检出的病毒含量无显著差异,分别为春季（2.69±1.46）×105 copies/g（消化腺）,夏季（1.97±2.16）×105 copies/g（消化腺）,秋季（6.91±6.16）×104 copies/g（消化腺）,冬季为（4.83±2.99）×104 copies/g（消化腺）。部分阳性样本测序分析后显示,除1份为GII.17基因型外,其余均为GII.4基因型（n=13）,与当地的临床流行基因型呈现一致性。本研究显示广州市售牡蛎中仍存在较高的诺如病毒污染水平,需要进一步加强病毒防控工作,尤其提醒消费者在食用牡蛎时需加工充分。     

Depuration of live oysters to reduce Vibrio parahaemolyticus and Vibrio vulnificus: A review of ecology and processing parameters

Consumption of raw oysters, whether wild-caught or aquacultured, may increase health risks for humans. Vibrio vulnificus and Vibrio parahaemolyticus are two potentially pathogenic bacteria that can be concentrated in oysters during filter feeding. As Vibrio abundance increases in coastal waters worldwide, ingesting raw oysters contaminated with V. vulnificus and V. parahaemolyticus can possibly result in human illness and death in susceptible individuals. Depuration is a postharvest processing method that maintains oyster viability while they filter clean salt water that either continuously flows through a holding tank or is recirculated and replenished periodically. This process can reduce endogenous bacteria, including coliforms, thus providing a safer, live oyster product for human consumption; however, depuration of Vibrios has presented challenges. When considering the difficulty of removing endogenous Vibrios in oysters, a more standardized framework of effective depuration parameters is needed. Understanding Vibrio ecology and its relation to certain depuration parameters could help optimize the process for the reduction of Vibrio. In the past, researchers have manipulated key depuration parameters like depuration processing time, water salinity, water temperature, and water flow rate and explored the use of processing additives to enhance disinfection in oysters. In summation, depuration processing from 4 to 6 days, low temperature, high salinity, and flowing water effectively reduced V. vulnificus and V. parahaemolyticus in live oysters. This review aims to emphasize trends among the results of these past works and provide suggestions for future oyster depuration studies.     

Fecal contamination and diarrheal pathogens on surfaces and in soils among Tanzanian households with and without improved sanitation

Little is known about the extent or pattern of environmental fecal contamination among households using low-cost, on-site sanitation facilities, or what role environmental contamination plays in the transmission of diarrheal disease. A microbial survey of fecal contamination and selected diarrheal pathogens in soil (n = 200), surface (n = 120), and produce samples (n = 24) was conducted in peri-urban Bagamoyo, Tanzania, among 20 households using private pit latrines. All samples were analyzed for E. coli and enterococci. A subset was analyzed for enterovirus, rotavirus, norovirus GI, norovirus GII, diarrheagenic E. coli, and general and human-specific Bacteroidales fecal markers using molecular methods. Soil collected from the house floor had significantly higher concentrations of E. coli and enterococci than soil collected from the latrine floor. There was no significant difference in fecal indicator bacteria levels between households using pit latrines with a concrete slab (improved sanitation) versus those without a slab. These findings imply that the presence of a concrete slab does not affect the level of fecal contamination in the household environment in this setting. Human Bacteroidales, pathogenic E. coli, enterovirus, and rotavirus genes were detected in soil samples, suggesting that soil should be given more attention as a transmission pathway of diarrheal illness in low-income countries.     

Effects of High Hydrostatic Pressure on the Physical,Microbial, and Chemical Attributes of Oysters (Crassostrea virginica)

The change in the quality attributes (physical, microbial, and chemical) of oysters (Crassostrea virginica) after high hydrostatic pressure (HHP) treatment at 300 MPa at room temperature (RT, 25 °C) 300, 450, and 500 MPa at 0 °C for 2 min and control oysters without treatment were evaluated over 3 wk. The texture and tissue yield percentages of oysters HHP treated at 300 MPa, RT increased significantly (P < 0.05) compared to control. Aerobic and psychrotrophic bacteria in control oysters reached the spoilage point of 7 log CFU/g after 15 d. Coliform counts (log MPN/g) were low during storage with total and fecal coliforms less than 3.5 and 1.0. High pressure treated oysters at 500 MPa at 0 °C were significantly higher (P < 0.05) than oysters HHP treated at 300 MPa at 0 °C in lipid oxidation values. The highest pressure (500 MPa) treatment in this study, significantly (P < 0.05) decreased unsaturated fatty acid percentage compared to control. The glycogen content of control oysters at 3 wk was significantly higher (P < 0.05) when compared to HHP treated oysters [300 MPa, (RT); 450 MPa (0 °C); and 500 MPa (0 °C)]. HHP treatments of oysters were not significantly different in pH, percent salt extractable protein (SEP), and total lipid values compared to control. Based on our results, HHP prolongs the physical, microbial, and chemical quality of oysters.     

Microbial characterization and population changes in nonpotable reclaimed water distribution systems

Changes in the microbial quality of nonpotable reclaimed water distribution systems in seven metropolitan areas of the southwestern United States were investigated by performing pathogen monitoring and bacterial growth. Water samples were collected from tertiary-treated effluents at point of entry and point of use in the distribution systems. The samples were analyzed for Cryptosporidium oocysts, Giardia cysts, enteroviruses, microbial indicators, and assimilable organic carbon (AOC). Cryptosporidium and Giardia were detected in 16% (12/77) and 43% (33/77) of nonpotable reclaimed water samples, whereas no infectious Cryptosporidium parvum were detected in any of the samples. No infectious enteroviruses were detected in any sample (0/27). At point of entry total coliform and fecal coliforms ranged from 0.7 to 280 and from 0 to 1.9 colony-forming units (cfu)/100 mL, respectively. Increases in the number of coliforms were observed as water passed through the reclaimed water distribution systems. However, no such increase in the number of coliphages was found. The chlorination practices at some utilities were not sufficient to inactivate coliforms and coliphages, but supplemental ultraviolet disinfection resulted in lower numbers of these microbial indicators. AOC levels decreased by 3-fold as water passed through the distribution systems, which inversely correlated with bacterial regrowth.     

Combined effect of ozonated water and chitosan on the shelf-life of Pacific oyster (Crassostrea gigas)

Effects of ozonated water and chitosan treatment on the shelf-life extension of Pacific oysters (Crassostrea gigas) stored at 5 ± 1 °C were studied. Results indicated that ozonated water treatment reduced the total microbial load of fresh oysters by about 10-fold (from 3.2 × 10³ CFU/g to 1.8 × 10² CFU/g) before storage and the microbial flora was different with that of raw samples. The wide-spectrum antibacterial property of chitosan against bacteria isolated from oysters was confirmed, and chitosan concentration of 5.0 g/l was eventually determined for application in oyster preservation. Based on microbiological analysis, biochemical indices determination and sensory evaluation, shelf-lives of 8–9 days for control, 10–12 days for ozonated water treated samples, 14–15 days for chitosan treated samples and 20–21 days for samples with combined treatment were observed, indicating that ozonated water and chitosan have a great potential for oyster preservation.Industrial relevanceAs seafood, Pacific oysters have a short shelf-life. Improvements in the shelf-life of oysters can have an important economic impact by reducing losses and by allowing the products to reach distant and new markets.In this work, Shelf-life of oysters with combined treatment of ozonated water and chitosan doubled, which has great practical meaning, and the process could be fully adopted by the food industry.We also did some research about the changes in microbial flora after ozonated water treatment. This work could help in preservation of oysters when ozone or ozonated water concerned.We discovered Wide-spectrum antibacterial property of chitosan against the strains isolated from raw oysters. The potential for using chitosan as a natural preservative in oysters was approved.     

Effects of high-pressure and heat treatments on physical and biochemical characteristics of oysters (Crassostrea gigas)

Physical and biochemical changes in oysters following high-pressure (HP) treatment at 260 MPa for 3 min or heat treatment (cool pasteurisation (CP) at 50 °C for 10 min or traditional pasteurisation (TP) at 75 °C for 8 min) were investigated and compared to changes in untreated oysters. HP or TP oysters had higher (P < 0.05) pH values (6.49–6.58) than untreated or CP oysters (6.45–6.46). HP and heat treatment both modified the gross composition of oyster tissue. The protein content of HP-treated oysters (6.9%) was significantly (P < 0.05) lower compared to control or heat-treated oysters (7.9–9.1%). The moisture content of HP-treated whole oyster tissue (86.5%) was higher than that of heat-treated or untreated oysters (83.5–84.7%), but HP or CP treatments did not affect the salt content or water activity of oysters. However, all treatments increased Hunter L- (66.3–68.9) while decreasing a- (− 1.6 to − 2.4) and b- (15.8–14.5) values of oyster tissue; overall, HP treatment had less negative effects on tissue colour of oysters than thermal treatments. HP-treated, CP and TP oysters had higher shucking yields (15.5%, 12.5% and 2.6%, respectively) than untreated oysters. One significant advantage of HP treatment over heat treatment of oysters was that the former process opened the oyster and separated the muscle of the oyster from the shell.     

季节性自回归差分移动平均模型在牡蛎中诺如病毒检出率预测上的应用

目的 基于季节性自回归差分移动平均(ARIMA)模型分析并预测上海市售牡蛎中诺如病毒(NoV)的检出率,为水产品中NoV的污染规律提供参考。方法 2016年6月—2019年11月,从上海芦潮港海鲜市场定期采购牡蛎样品共531只,通过巢式聚合酶链式反应(Nest-PCR),对其进行了NoV检测,按季度分析检出率。采用季节性ARIMA模型对牡蛎中NoV的检出率数据拟合建立模型,经过数据平稳化、模型选择和拟合、模型诊断得到最优模型,并运用最优模型对未来四个季度牡蛎中NoV的检出率进行预测。结果 拟合出的季节性ARIMA(0,1,1)(0,1,0)₄为最优模型,赤池信息量准则的修正值(AICc)最小(58.70),残差经Ljung-Box检验为白噪声序列。模型拟合牡蛎中NoV的阳性率趋势与实际检出率趋势基本吻合,平均绝对误差(MAE)为4.85,平均绝对百分比误差(MAPE)为30.25。用最优模型预测的未来四个季度牡蛎中NoV阳性检出率分别为31.89%、12.80%、9.47%、6.14%。结论 季节性ARIMA模型(0,1,1)(0,1,0)₄能较好地拟合牡蛎中NoV的阳性检出率趋势,对NoV污染的牡蛎等水产品的风险评估及NoV流行的防控具有一定的意义。     

Detection of noroviruses in shellfish in the Netherlands

Shellfish from oyster farms in the Netherlands and imported from other European countries were examined for viral contamination. A method that allows sequence matching between noroviruses from human cases and shellfish was used. The samples of shellfish (n = 42) were analyzed using a semi-nested RT-PCR that had been optimized for detection of norovirus in shellfish (SR primer sets). In addition, a different genome region was targeted using a second primer set which is routinely used for diagnosis of norovirus infection in humans (JV12Y/JV13I). To improve the detection limit for this RT-PCR a semi-nested test format was developed (NV primer sets). One of 21 oyster samples (4.8%) from Dutch farms was norovirus positive, whereas norovirus was detected in 1 out of 8 oyster samples (12.5%) and 5 out of 13 mussel samples (38.5%) collected directly after importation in the Netherlands. RNA from samples associated with an outbreak of gastro-enteritis in the Netherlands in 2001 was re-analyzed using the NV primer sets. At least one identical sequence (142/142 nt) was found in three fecal and in two oyster samples related to this outbreak. Further surveillance of norovirus by detection and typing of viruses from patients with gastroenteritis and shellfish is warranted to clarify the causes of future outbreaks.     

Fatty acids,volatile compounds and colour changes in high-pressure-treated oysters (Crassostrea gigas)

Proximal composition, colour and levels of fatty acids and volatile compounds in oysters following high-pressure (HP) treatment at 260, 500 or 800 MPa for 3, 5 or 5 min, respectively, were investigated and compared to untreated oysters. HP-treated oysters had significantly (P < 0.05) higher pH than untreated oysters. HP treatment also modified the gross composition of oyster tissue, the moisture content of HP-treated oyster being higher than that of untreated oyster. HP treatment at 260 MPa had less negative effects on oyster tissue colour (Hunter L-, a- and b-values) than treatment at higher pressures. HP treatment of oysters resulted in no significant changes in the fatty acid profile compared to untreated oysters. HP treatment of oysters, however, changed the level of volatile components when compared to the headspace of fresh oysters; HP-treated samples had higher concentrations of dimethyl sulfide, 1-penten-3-one, phenol and 1,2,4-trimethylbenzene relative to untreated samples. On the other hand, HP-treated oysters had lower concentrations of 1-penten-3-ol, 2,3-pentanedione, (E,E,Z)-1,3,5-octatriene and 1,3-octadiene than untreated samples.Industrial relevanceHigh-pressure (HP) treatment is being increasingly employed for commercial processing of oysters. While the profiles of fatty acids and volatiles of fresh oysters have been extensively studied, no studies reporting the effects of HP on these have been reported. Compared to fresh oysters, HP treatment did not change significantly the fatty acid profile; however, differences in the headspace volatile profile of HP-treated oysters were noticed.     

Demonstration of Indicator Microorganisms on Surface of Vegetables on the Market in the United States and Mexico

ABSTRACT: Presence of indicator microorganisms of fecal pollution on the surface of vegetables sold in retail markets is demonstrated. Vegetables were obtained from local markets in the U.S.A. and Mexico. At least 1 of the indicators, among coliphages, fecal streptococci, total coliforms, and fecal coliforms, was detected in every sample tested. Relatively small amounts of indicators were recovered from the U.S.A. samples. Fecal streptococci showed significant correlations with all other indicators. However, some U.S.A. samples low in fecal streptococci were found to harbor certain levels of other indicators. Upon introduction of an indicator system to detect the fecal contamination of agriculture crops, it is recommended that multiple indicator microorganisms be measured.     

Norovirus in retail shellfish

Norovirus is a common cause of gastroenteritis outbreaks associated with consumption of raw shellfish. The majority of norovirus infections worldwide are due to genogroup II noroviruses. Bivalve molluscs (mussels, clams and oysters) at the end of the commercial chain, the points of purchase, were sampled between 2005 and 2008 in several retail points in Apulia, Italy, and screened by a semi-nested RT-PCR specific for genogroup II noroviruses. Noroviral RNA was detected in 12.1% of the samples, with lower frequency being observed in samples obtained from hypermarkets (8.1%) rather than in samples from open-air markets and fish shops (17.6% and 16.2%, respectively). By sequence analysis, the strains were characterized as norovirus variants GII.4/2004 and GII.b/Hilversum, which were both circulating in Italy in the same time-span.     

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.