Risk Assessment of Norovirus Illness from Consumption of Raw Oysters in the United States and in Canada.

Human norovirus (NoV) is the leading cause of foodborne illness in the United States and Canada. Bivalve molluscan shellfish is one commodity commonly identified as being a vector of NoV. Bivalve molluscan shellfish are grown in waters that may be affected by contamination events, tend to bioaccumulate viruses, and are frequently eaten raw. In an effort to better assess the elements that contribute to potential risk of NoV infection and illness from consumption of bivalve molluscan shellfish, the U.S. Department of Health and Human Services/Food and Drug Administration (FDA), Health Canada (HC), the Canadian Food Inspection Agency (CFIA), and Environment and Climate Change Canada (ECCC) collaborated to conduct a quantitative risk assessment for NoV in bivalve molluscan shellfish, notably oysters. This study describes the model and scenarios developed and results obtained to assess the risk of NoV infection and illness from consumption of raw oysters harvested from a quasi-steady-state situation. Among the many factors that influence the risk of NoV illness for raw oyster consumers, the concentrations of NoV in the influent (raw, untreated) and effluent (treated) of wastewater treatment plants (WWTP) were identified to be the most important. Thus, mitigation and control strategies that limit the influence from human waste (WWTP outfalls) in oyster growing areas have a major influence on the risk of illness from consumption of those oysters.

Detection and molecular characterization of norovirus from oysters implicated in outbreaks in the US.

Human noroviruses are the leading cause of non-bacterial shellfish associated gastroenteritis. Here we report on the detection and characterization of norovirus (NoV) in shellfish associated outbreaks. Requests were received from state and federal officials for technical assistance in the analysis of shellfish for NoV and male specific coliphage (MSC; an enteric virus surrogate) during the years 2009 thru 2014. In outbreaks where NoV was detected, genogroup II (GII) levels ranged from 2.4 to 82.0 RT-qPCR U/g of digestive diverticula (DD) while NoV genogroup I (GI) levels ranged from 1.5 to 29.8 RT-qPCR U/g of DD. Murine norovirus extraction efficiencies ranged between 50 and 85%. MSC levels ranged from <6 to 80 PFU/100 g. Phylogenetic analysis of the outbreak sequences revealed strains clustering with GI.8, GI.4, GII.3, GII.4, GII.7, and GII.21. There was 100% homology between the shellfish and clinical strains occurring in 2 of 8 outbreaks. Known shellfish consumption data demonstrated probable infectious particles ingested as low as 12. These investigations demonstrate effective detection, quantification, and characterization of NoV in shellfish associated with illness.

Meta-Analysis of the Reduction of Norovirus and Male-Specific Coliphage Concentrations in Wastewater Treatment Plants.

Human norovirus (NoV) is the leading cause of foodborne illness in the United States and Canada. Wastewater treatment plant (WWTP) effluents impacting bivalve mollusk-growing areas are potential sources of NoV contamination. We have developed a meta-analysis that evaluates WWTP influent concentrations and log10 reductions of NoV genotype I (NoV GI; in numbers of genome copies per liter [gc/liter]), NoV genotype II (NoV GII; in gc/liter), and male-specific coliphage (MSC; in number of PFU per liter), a proposed viral surrogate for NoV. The meta-analysis included relevant data (2,943 measurements) reported in the scientific literature through September 2013 and previously unpublished surveillance data from the United States and Canada. Model results indicated that the mean WWTP influent concentration of NoV GII (3.9 log10 gc/liter; 95% credible interval [CI], 3.5, 4.3 log10 gc/liter) is larger than the value for NoV GI (1.5 log10 gc/liter; 95% CI, 0.4, 2.4 log10 gc/liter), with large variations occurring from one WWTP to another. For WWTPs with mechanical systems and chlorine disinfection, mean log10 reductions were -2.4 log10 gc/liter (95% CI, -3.9, -1.1 log10 gc/liter) for NoV GI, -2.7 log10 gc/liter (95% CI, -3.6, -1.9 log10 gc/liter) for NoV GII, and -2.9 log10 PFU per liter (95% CI, -3.4, -2.4 log10 PFU per liter) for MSCs. Comparable values for WWTPs with lagoon systems and chlorine disinfection were -1.4 log10 gc/liter (95% CI, -3.3, 0.5 log10 gc/liter) for NoV GI, -1.7 log10 gc/liter (95% CI, -3.1, -0.3 log10 gc/liter) for NoV GII, and -3.6 log10 PFU per liter (95% CI, -4.8, -2.4 PFU per liter) for MSCs. Within WWTPs, correlations exist between mean NoV GI and NoV GII influent concentrations and between the mean log10 reduction in NoV GII and the mean log10 reduction in MSCs.

Importance of histamine-producing Clostridium perfringens in scombrotoxin-forming fish.

It has been suggested that anaerobic histamine-producing bacteria (HPB) are important contributors to scombrotoxin fish poisoning (SFP). In order to assess the role of Clostridium perfringens in SFP, we developed a real-time PCR method for rapid detection of histamine-producing (HP) C. perfringens. The real-time PCR assay was 100% inclusive for detecting 23 HP C. perfringens and did not detect any of the other 116 HP or non-HP isolates examined. The efficiency of the assay with or without internal amplification control DNA was 102%; in the presence of background flora and inhibitory matrices, it was 90 to 99%. To investigate the importance of HP C. perfringens in SFP, we examined histamine production by C. perfringens in inoculated fish samples incubated under anaerobic conditions. C. perfringens produced low histamine levels in tuna (19 ppm) and Spanish mackerel (3 ppm), whereas gram-negative HPB produced high histamine levels (6,345 ppm in tuna; 1,223 ppm in Spanish mackerel) under the same conditions. When one bonito, two bigeye tuna, nine mahi-mahi, and five yellowfin tuna were examined for the presence of HPB, none (0 of 17) of the samples contained HP C. perfringens or other gram-positive HPB, whereas 86% of the samples contained gram-negative HPB. Our study indicates that histamine production by C. perfringens in scombrotoxin-forming fish was minimal compared with that by gram-negative HPB and that C. perfringens may not be an important bacterial species associated with SFP.

Surface plasmon resonance biosensor for detection of feline calicivirus, a surrogate for norovirus.

The human noroviruses are the most common non-bacterial cause of gastroenteritis and are responsible for as much as 50% of all gastroenteritis outbreaks worldwide. Norovirus (NoV), a single stranded RNA virus, is highly contagious with an infectious dose of less than 100 viral particles. While techniques exist for the identification of NoV, the lack of a reliable cell culture system, NoV genetic variability, and time-consuming sample preparation steps required to isolate the virus (or its genome) prior to molecular based methods has hindered rapid virus detection. To better protect the public from virus-contaminated food and enable better detection in clinical and environmental samples, sensitive and selective methods with simple sample preparation are needed. Surface plasmon resonance (SPR) biosensors represent an emerging detection platform, and this approach has been applied to the rapid detection of foodborne small molecule toxins, protein toxins, and bacteria. This analytical technique, however, has yet to be fully investigated for rapid virus detection, especially for intact viral particles extracted from food matrices. For this study, the culturable, non-human pathogen feline calicivirus (FCV), which has similar morphology and is genetically related to NoV, was chosen as a surrogate virus for designing and evaluating an SPR assay. An antibody-based assay was performed by first immobilizing anti-FCV to an SPR chip surface and then directly measuring virus binding and subsequent secondary antibody binding. The resulting biosensor directly detected intact FCV particles with limits of detection of approximately 10(4)TCID50FCV/mL from purified cell culture lysates. In addition, intact virus detection in FCV-spiked oyster matrix was possible when using a simple extraction procedure and employing a secondary antibody to FCV for quantitation. The results from these preliminary studies show promise for the development of a rapid assay for detecting intact viruses, such as NoV, using an SPR biosensor. While the current level of sensitivity achieved with this SPR biosensor may be more applicable to virus detection in clinical specimens, broader application and increased sensitivity of this method for foodborne viruses may be achieved when performed in conjunction with efficient virus extraction and concentration methods.

Seasonal levels of the Vibrio predator bacteriovorax in atlantic, pacific, and gulf coast seawater.

Bacteriovorax were quantified in US Atlantic, Gulf, and Pacific seawater to determine baseline levels of these predatory bacteria and possible seasonal fluctuations in levels. Surface seawater was analyzed monthly for 1 year from Kailua-Kona, Hawaii; the Gulf Coast of Alabama; and four sites along the Delaware Bay. Screening for Bacteriovorax was performed on lawns of V. parahaemolyticus host cells. Direct testing of 7.5 mL portions of seawater from the Atlantic, Pacific, and Gulf coasts gave mean annual counts ≤12.2 PFU. Spikes in counts were observed at 3 out of 4 sites along the Delaware Bay 1 week after Hurricane Sandy. A comparison of summer versus winter counts showed significantly more Bacteriovorax (P ≤ 0.0001) in the Delaware Bay during the summer and significantly more (P ≤ 0.0001) in the Gulf during the winter, but no significant seasonal differences (P > 0.05) for Hawaiian seawater. Bacteriovorax counts only correlated with seawater salinity and temperature at one Delaware site (r = 0.79 and r = 0.65, resp.). There was a relatively strong negative correlation between temperature and Bacteriovorax levels (r = -0.585) for Gulf seawater. Selected isolates were sequenced and identified by phylogenetic analysis as Bacteriovorax clusters IX, X, XI, and XII.

Direct sequencing of hepatitis A virus and norovirus RT-PCR products from environmentally contaminated oyster using M13-tailed primers.

Human norovirus (HuNoV) and hepatitis A (HAV) are recognized as leading causes of non-bacterial foodborne associated illnesses in the United States. DNA sequencing is generally considered the standard for accurate viral genotyping in support of epidemiological investigations. Due to the genetic diversity of noroviruses (NoV), degenerate primer sets are often used in conventional reverse transcription (RT) PCR and real-time RT-quantitative PCR (RT-qPCR) for the detection of these viruses and cDNA fragments are generally cloned prior to sequencing. HAV detection methods that are sensitive and specific for real-time RT-qPCR yields small fragments sizes of 89-150bp, which can be difficult to sequence. In order to overcome these obstacles, norovirus and HAV primers were tailed with M13 forward and reverse primers. This modification increases the sequenced product size and allows for direct sequencing of the amplicons utilizing complementary M13 primers. HuNoV and HAV cDNA products from environmentally contaminated oysters were analyzed using this method. Alignments of the sequenced samples revealed ≥95% nucleotide identities. Tailing NoV and HAV primers with M13 sequence increases the cDNA product size, offers an alternative to cloning, and allows for rapid, accurate and direct sequencing of cDNA products produced by conventional or real time RT-qPCR assays.

Quantification of total and specific gram-negative histamine-producing bacteria species in fish using an MPN real-time PCR method.

Quantification of histamine-producing bacteria (HPB) is necessary in order to elucidate the role that HPB play in scombrotoxin (histamine) fish poisoning. We report here the evaluation of a real-time PCR method for the quantification of total and specific Gram-negative HPB species in fish using a most probable number (MPN) format. The species-specific real-time PCR assay was 100% inclusive for independently detecting Morganella morganii, Enterobacter aerogenes, Raoultella planticola/ornithinolytica and Photobacterium damselae and did not cross react with other histamine- or non- histamine-producing bacteria. The efficiency of the reactions in the absence and presence of Spanish mackerel enrichment containing 1 × 10(6) CFU/ml of background microflora were 93-104 and 92-99%, respectively. The MPN-real-time PCR assay accurately quantified total and specific HPB in spiked mahi-mahi (Coryphaena hippurus) and Spanish mackerel (Scomberomorus maculates) samples. These methods were used to quantify total and specific HPB in naturally contaminated, decomposing mahi-mahi, Spanish mackerel and tuna (Thunnus albacares) samples. The results of this study indicate that MPN-real-time PCR assays can be used to accurately enumerate total and specific HPB in fish samples. These assays can be applied to assess the effectiveness of mitigation strategies and understand the relationship between HPB and histamine production in decomposing fish.

Development of a real-time PCR assay with an internal amplification control for detection of Gram-negative histamine-producing bacteria in fish.

Prompt detection of bacteria that contribute to scombrotoxin (histamine) fish poisoning can aid in the detection of potentially toxic fish products and prevent the occurrence of illness. We report development of the first real-time PCR method for rapid detection of Gram-negative histamine-producing bacteria (HPB) in fish. The real-time PCR assay was 100% inclusive for detecting high-histamine producing isolates and did not detect any of the low- or non-histamine producing isolates. The efficiency of the assay with/without internal amplification control ranged from 96-104% and in the presence of background flora and inhibitory matrices was 92/100% and 73-96%, respectively. This assay was used to detect HPB from naturally contaminated yellowfin tuna, bluefish, and false albacore samples. Photobacterium damselae (8), Plesiomonas shigelloides (2), Shewanella sp. (1), and Morganella morganii (1) were subsequently isolated from the real-time PCR positive fish samples. These results indicate that the real-time PCR assay developed in this study is a rapid and sensitive method for detecting high-HPB. The assay may be adapted for quantification of HPB, either directly or with an MPN-PCR method.

Bacterial and viral pathogens in live oysters: 2007 United States market survey.

Two samples of market oysters, primarily from retail establishments, were collected twice each month in each of nine states during 2007. Samples were shipped refrigerated overnight to five U.S. Food and Drug Administration laboratories on a rotating basis and analyzed by most probable number (MPN) for total and pathogenic Vibrio parahaemolyticus and V. vulnificus numbers and for the presence of toxigenic V. cholerae, Salmonella spp., norovirus (NoV), and hepatitis A virus (HAV). Levels of indicator organisms, including fecal coliforms (MPN), Escherichia coli (MPN), male-specific bacteriophage, and aerobic plate counts, were also determined. V. parahaemolyticus and V. vulnificus levels were distributed seasonally and geographically by harvest region and were similar to levels observed in a previous study conducted in 1998-1999. Levels of pathogenic V. parahaemolyticus were typically several logs lower than total V. parahaemolyticus levels regardless of season or region. Pathogenic V. parahaemolyticus levels in the Gulf and Mid-Atlantic regions were about two logs greater than the levels observed in the Pacific and North Atlantic regions. Pathogens generally associated with fecal pollution were detected sporadically or not at all (toxigenic V. cholerae, 0%; Salmonella, 1.5%; NoV, 3.9%; HAV, 4.4%). While seasonal prevalences of NoV and HAV were generally greater in oysters harvested from December to March, the low detection frequency obscured any apparent seasonal effects. Overall, there was no relationship between the levels of indicator microorganisms and the presence of enteric viruses. These data provide a baseline that can be used to further validate risk assessment predictions, determine the effectiveness of new control measures, and compare the level of protection provided by the U.S. shellfish sanitation system to those in other countries.

Use of N stable isotope and microbial analyses to define wastewater influence in Mobile Bay, AL.

We assessed short-term ecological and potential human health effects of wastewater treatment plant (WTP) effluent by measuring delta 15N per thousand and microbial concentrations in oysters and suspended particulate matter (SPM). We also tested male-specific bacteriophage (MSB) as an alternative to fecal coliforms, to assess potential influence of wastewater contamination on shellfish. WTP effluent did not affect oyster growth or survival, but SPM and oysters acquired wastewater-specific delta 15N per thousand. delta 15N values were depleted near the WTP, typical of low-level processed wastewater. Fecal coliform and MSB concentrations were higher in samples taken closest to the WTP, and MSB values were significantly correlated with delta 15N per thousand in oyster tissues. Overall, oysters demonstrated relatively rapid integration and accumulation of wastewater-specific delta 15N per thousand and indicator microorganisms compared to water samples. These data suggest oysters were superior sentinels compared to water, and MSB was a more reliable indicator of wastewater influence on shellfish than fecal coliforms.

Salmonella and the sanitary quality of aquacultured shrimp.

In this study, we examined the prevalence of Salmonella and coliform bacteria on shrimp aquaculture farms to develop guidelines or preventative measures for reducing Salmonella and fecal contamination on products harvested from these farms. The U.S. Food and Drug Administration, in conjunction with foreign government regulatory agencies, the aquaculture industry, and academia affiliates, analyzed 1,234 samples from 103 shrimp aquaculture farms representing six countries between July 2001 and June 2003 for fecal coliforms, Escherichia coli, and Salmonella. A significant relationship was found (P = 0.0342) between the log number of fecal bacteria and the probability that any given sample would contain Salmonella. The likelihood of any given sample containing Salmonella was increased by 1.2 times with each 10-fold increase in either fecal coliform or E. coli concentration. The statistical relationship between Salmonella concentration and that of both fecal coliforms and E. coli was highest in grow-out pond water (P = 0.0042 for fecal coliforms and P = 0.0021 for E. coli). The likelihood of finding Salmonella in grow-out pond water increased 2.7 times with each log unit increase in fecal coliform concentration and 3.0 times with each log unit increase in E. coli concentration. Salmonella is not part of the natural flora of the shrimp culture environment nor is it inherently present in shrimp grow-out ponds. The occurrence of Salmonella bacteria in shrimp from aquaculture operations is related to the concentration of fecal bacteria in the source and grow-out pond water.