Comparison of norovirus RNA levels in outbreak-related oysters with background environmental levels

Norovirus is the principal agent of bivalve shellfish-associated gastroenteric illness worldwide. Numerous studies using PCR have demonstrated norovirus contamination in a significant proportion of both oyster and other bivalve shellfish production areas and ready-to-eat products. By comparison, the number of epidemiologically confirmed shellfish-associated outbreaks is relatively low. This suggests that factors other than the simple presence or absence of virus RNA are important contributors to the amount of illness reported. This study compares norovirus RNA levels in oyster samples strongly linked to norovirus or norovirus-type illness with the levels typically found in commercial production areas (non-outbreak-related samples). A statistically significant difference between norovirus levels in the two sets of samples was observed. The geometric mean of the levels in outbreak samples (1,048 copies per g) was almost one order of magnitude higher than for positive non-outbreak-related samples (121 copies per g). Further, while none of the outbreak-related samples contained fewer than 152 copies per g, the majority of positive results for non-outbreak-related samples was below this level. These observations support the concept of a dose-response for norovirus RNA levels in shellfish and could help inform the establishment of threshold criteria for risk management.     

Multiple Clusters of Norovirus among Shellfish Consumers Linked to Symptomatic Oyster Harvesters

We describe the investigation of a norovirus outbreak associated with raw oyster consumption affecting 36 people in British Columbia, Canada, in 2010. Several genotypes were found in oysters, including an exact sequence match to clinical samples in regions B and C of the norovirus genome (genogroup I genotype 4). Traceback implicated a single remotely located harvest site probably contaminated by ill shellfish workers during harvesting activities. This outbreak resulted in three recalls, one public advisory, and closure of the harvest site.     

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.     

A semiquantitative approach to estimate Norwalk-like virus contamination of oysters implicated in an outbreak

Gastroenteritis outbreaks linked to shellfish consumption are numerous and Norwalk-like viruses (NLVs) are frequently the responsible causative agents. However, molecular data linking shellfish and clinical samples are still rare despite the availability of diagnostic methods. In a recent outbreak we found the same NLV sequence in stool and shellfish samples (100% identity over 313 bp in the capsid region), supporting the epidemiological data implicating the shellfish as the source of infection. A semiquantitative approach using most-probable-number-RT-PCR (MPN-RT-PCR) demonstrated the presence of a hundred of RT-PCR units per oyster. Follow-up of the oysters in the harvest area, for approximately 2 months, showed persistence of NLV contamination of the shellfish at levels up to a thousand RT-PCR units per oyster prior to depuration of the shellfish. This finding is useful in beginning to understand shellfish contamination and depuration for use in future hazard analyses.     

Detection of norovirus RNA in bivalve molluscs by using bacteria-culture-employed method (A3T method)

Norovirus (NV) RNA has rarely been detected in foods despite the use of highly sensitive methods such as RT-PCR and real-time RT-PCR. In the modified method (A3T method) reported previously, a bacterial culture process was introduced into the standard protocol for NV detection to remove some inhibitor(s) present in food ingredients. To confirm the efficiency of the A3T method and to examine NV contamination in bivalve molluscs, we tried to detect NV RNA in bivalve molluscs on the market and in oyster samples associated with foodborne outbreaks by using the standard method and the A3T method. NV RNAs were detected in 20 samples (18.0%) of 111 bivalve molluscs, including oysters, on the market by use of the A3T method, while only one sample (0.9%) was positive according to the standard method. NV RNA was also detected in 10 of 35 oyster samples related to foodborne outbreaks by the A3T method. Those results show that the A3T method is suitable for the detection of NV in bivalve molluscs in general laboratories.     

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。     

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...     

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.     

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.     

Detection of histamine-producing bacteria using polymerase chain reaction techniques and DNA probes

The polymerase chain reaction technique was used to enable the detection of DNA specific amplicons, corresponding to gene fragments coding for histidine decarboxylase present in the histidine decarboxylating bacteria frequently found in canned fish. The level of histamine in several fish product samples was quantified by HPLC and the DNA of the samples containing histamine was isolated and amplified with hdcA histidine-decarboxylase gene-specific primers. The primer sets used, CL1/CL2, JV16HC/JV17HC and CL1/JV17HC, amplified 150 bp, 370 bp and 500 bp products respectively. Non-expected fragments (100 bp, 150 bp and 250 bp) were also amplified by JV16HC/JV17HC. Some amplified fragments were sequenced automatically, presenting high homologies with the Clostridium perfringens hdcA gene. A DNA probe from a C. perfringens pure strain was hybridized with the DNA fragments amplified from the contaminated samples. The hybridization blots were then detected by colorimetry, revealing that the samples were contaminated by C. perfringens.     

河北省市售贝类中脂溶性毒素的调查研究

目的调查河北省市售贝类中脂溶性贝类毒素的污染状况。方法在2018年8月—2020年4月间,对河北省市售7大类贝类样品进行采集,共354份,采用超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)进行脂溶性贝类毒素的检测。结果 354份样品中,阳性样品17份,检出率为4.8%,检出含量较低,检出品种有毛蚶、贻贝、扇贝、牡蛎,毒素组分有虾夷扇贝毒素(yessotoxin,YTX)、同源虾夷扇贝毒素(homo-YTX)、大田软海绵酸(okadaicacid,OA)、鳍藻毒素(dinophysistoxins, DTXs)(DTX1、DTX2)、蛤毒素2(pectenotoxin-2, PTX2)。结论河北省市售贝类中脂溶性贝类毒素暴露风险很低。