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.

Model results indicated that the mean WWTP influent concentration of NoV GII (3.9 log 10 gc/liter; 95% credible interval [CI], 3.5, 4.3 log 10 gc/liter) is larger than the value for NoV GI (1.5 log 10 gc/liter; 95% CI, 0.4, 2.4 log 10 gc/liter), with large variations occurring from one WWTP to another. For WWTPs with mechanical systems and chlorine disinfection, mean log 10 reductions were ؊2.4 log 10 gc/liter (95% CI, ؊3.9, ؊1.1 log 10 gc/liter) for NoV GI, ؊2.7 log 10 gc/liter (95% CI, ؊3.6, ؊1.9 log 10 gc/liter) for NoV GII, and ؊2.9 log 10 PFU per liter (95% CI, ؊3.4, ؊2.4 log 10 PFU per liter) for MSCs. Comparable values for WWTPs with lagoon systems and chlorine disinfection were ؊1.4 log 10 gc/liter (95% CI, ؊3.3, 0.5 log 10 gc/liter) for NoV GI, ؊1.7 log 10 gc/liter (95% CI, ؊3.1, ؊0.3 log 10 gc/liter) for NoV GII, and ؊3.6 log 10 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 log 10 reduction in NoV GII and the mean log 10 reduction in MSCs.H uman norovirus (NoV) is the leading cause of food-associated gastroenteritis in the United States (1) and Canada (2). U.S. residents are estimated to experience five episodes of norovirus gastroenteritis in their lifetimes (3). NoV is primarily spread via the fecal-oral route. However, attribution of a particular case of NoV illness to a specific source is complex. The transmission may be direct (person to person) or indirect (via contact with contaminated fomites) or may occur through the ingestion of contaminated food or water (4). Noroviruses are genetically diverse, comprising six genogroups (5), three of which (genogroup I [GI], GII, and GIV) are capable of causing illness in humans (6).Among foodborne NoV outbreaks, bivalve molluscs (e.g., clams, oysters, mussels), leafy vegetables, and fruits are the most frequently implicated (7). More than half of the norovirus outbreaks attributed to the consumption of bivalve molluscs in the United States during the years from 2001 to 2008 are believed to have originated from contamination during production or processing (7). Bivalve molluscan shellfish typically grow in estuaries, which may contain NoV-contaminated human fecal material from municipal wastewater outfalls, combined sewer overflow, or nonpoint sources of pollution, including human waste discharged from marine vessels (8, 9). Bivalve molluscan shellfish feed on algae from the surrounding water. During this feeding process, each bivalve mollusc may filter 20 to 90 liters of water per day and bioaccumulate a variety of microorganisms, including viruses and bacteria that are associated with pollution sources (8,(10)(11)(12). Significantly, molluscan shellfish have been found to retain viruses to a greater extent and for much longer periods than they do bacteria (8,13,14). Bivalve molluscs, therefore, may become contaminated with NoV when they are grown in harvesting areas contaminated with human wastes.In the United States and in Canada, ar...

Aims:  To characterize the specificity and effect of pH and ionic strength on the kinetics of virus binding to histo‐blood group antigens (HBGA)‐conjugated magnetic beads. Methods and Results:  HBGAs from porcine gastric mucin (PGM) have been conjugated to magnetic beads (PGM‐MB) for concentration of NoV. A GII.4 virus was used for the detailed binding kinetics study and a panel of genogroup I (GI) NoVs, genogroup II (GII) NoVs and recombinant NoVs (rNoVs) were used for specificity and binding efficiency assays. We determined that NoV can be captured after 15 min of incubation with PGM‐MB, and virus recovery efficiency is decreased after extended incubation times. rNoV binding as measured by ELISA and NoV recovery as measured by quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR), were both enhanced significantly at acidic pH conditions. rNoV binding to PGM as measured by ELISA was increased up to 66%. While real‐time RT‐PCR analyses suggest that NoV could be concentrated as much as 1000‐fold at neutral pH, up to 3·4‐fold further increase of NoV recovery was achieved by adjusting the pH of the sample to 3·0–4·2. Variation between GI and GII viral binding to the PGM‐MB at basic pH was observed. All five GI rNoVs tested and 6 of 9 GII rNoVs were captured by PGM. All eight GI strains tested were concentrated by PGM‐MB, ranging from 28‐fold (GI.4) to 1502‐fold (GI.1). Eleven of 13 GII strains were concentrated from 30‐fold (GII.5) to 1014‐fold (GII.4, lab strain) by PGM‐MB. GI and GII rNoVs viral capsid proteins were recovered with high salt conditions, but results were inconsistent for whole virus recovery. Conclusions:  All GI and 85% of GII NoVs tested could be captured and concentrated by PGM‐MB method. The binding occurred rapidly and was enhanced at low pH. Significance and Impact of the Study:  These results facilitated development of a prototype method for sensitive detection of NoV in samples requiring larger volumes.

Numerous hepatitis A outbreaks were linked to the consumption of raw molluscan shellfish in the United States between 1960 and 1989. However, there had been no major molluscan shellfish-associated hepatitis A outbreaks reported in the United States for more than a decade (1989 to 2004). Beginning in late August 2005, at least 10 clusters of hepatitis A illnesses, totaling 39 persons, occurred in four states among restaurant patrons who ate oysters. Epidemiologic data indicated that oysters were the source of the outbreak. Traceback information showed that the implicated oysters were harvested from specific Gulf Coast areas. A voluntary recall of oysters was initiated in September. Hepatitis A virus (HAV) was detected in multiple 25-g portions in one of two recalled samples, indicating that as many as 1 of every 15 oysters from this source was contaminated. Comparing 315 nucleotides within the HAV VPl-2B region, 100% homology was found among four amplicons recovered from a total of six independent experiments of the implicated oysters, and an identical HAV sequence was detected in sera from all 28 patient serum specimens tested. Ten percent heterogeneity over 315 nucleotides (31 variants) was observed between the outbreak strain (subgenotype 1A) and an HM-175 strain (subgenotype 1B) used in the laboratory where the oysters were processed. To our knowledge, this investigation is the first in the United States to identify an HAV-identical strain in persons with hepatitis A as well as in the food that was implicated as the source of their infections.

An 18-month survey was conducted to examine the prevalence of enteric viruses and their relationship to indicators in environmentally polluted shellfish. Groups of oysters, one group per 4 weeks, were relocated to a coastal water area in the Gulf of Mexico that is impacted by municipal sewage and were analyzed for enteroviruses, Norwalk-like viruses (NLV), and indicator microorganisms (fecal coliform, Escherichia coli, and male-specific coliphages). The levels of indicator microorganisms were consistent with the expected continuous pollution of the area. Fourteen of the 18 oyster samples were found by reverse transcription (RT)-PCR to harbor NLV and/or enterovirus sequences. Of the four virus-negative oysters, three had exposure to water temperatures of >29°C. Concomitant with these findings, two of these four oysters also accumulated the lowest levels of coliphages. PCR primers targeting pan-enteroviruses and the NLV 95/96-US common subset were utilized; NLV sequences were detected more frequently than those of enteroviruses. Within the 12-month sampling period, NLV and enterovirus sequences were detected in 58 and 42%, respectively, of the oysters (67% of the oysters tested were positive for at least one virus) from a prohibited shellfish-growing area approximately 30 m away from a sewage discharge site. Eight (4.6%) of the 175 NLV capsid nucleotide sequences were heterogeneous among the clones derived from naturally polluted oysters. Overall, enteric viral sequences were found in the contaminated oysters throughout all seasons except hot summer, with a higher prevalence of NLV than enterovirus. Although a high percentage of the oysters harbored enteric viruses, the virus levels were usually less than or equal to 2 logs of RT-PCR-detectable units per gram of oyster meat.Virus infection accounts for two-thirds of the 13.8 million food-borne illnesses each year in the United States for which the pathogen is known (25). Major food vehicles associated with viral gastrointestinal diseases are shellfish (12,21,30,31), fresh produce and produce products (17,28,32), and readyto-eat deli food (1). Shellfish and fresh produce have been fecally contaminated in the field before or during harvest. Illegal overboard sewage discharges into shellfish harvesting waters was the most probable cause for recent major U.S. outbreaks (4, 37). Norwalk-like virus (NLV), recently renamed norovirus, was largely responsible for U.S. shellfish-borne viral gastroenteritis in the 1990s (8,19,22,35) and has been classified into different genogroups (16,39). Subgrouping of NLV strains has been carried out based upon (i) amino acid sequences of the open-reading frame 2 capsid region (2), resulting in 15 genetic clusters for genogroups I and II, (ii) the NLV capsid N/S domain (18), etc. Importantly, NLV genogroup II (among four genogroups) was identified predominantly in the 1990s outbreaks (10,11,13,20,24,41), and the 95/96-US subset of genotype II (cluster 4) was responsible for many outbreaks in the U.S. and elsewhere (15,29,40,41).Successfu...

Porphyromonas gingivalis is a key periodontal pathogen that has been implicated in the aetiology of chronic adult periodontitis. The aim of this study was to characterize two potential vaccine candidates (PG32 and PG33) identified from a previous genomic sequence analysis. Gene knockout studies suggested that these proteins play an important role in bacterial growth and are transcriptionally linked. Analysis of 14 laboratory and clinical isolates of P. gingivalis found that in all strains, both genes were present with a high level of conservation and that the two proteins were also expressed in vitro. Truncated recombinant PG32 and PG33 proteins were produced in Escherichia coli in an attempt to increase the solubility of the proteins while retaining their native conformation. While most of the truncated proteins remained insoluble, two truncated proteins showed good solubility and high levels of protection in the P. gingivalis murine lesion model and may be considered as potential vaccine candidates for further testing in models of human periodontal disease.

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During the summer of 2016, the Hawaii Department of Health responded to the second-largest domestic foodborne hepatitis A virus (HAV) outbreak in the post-vaccine era. The epidemiological investigation included case finding and investigation, sequencing of RNA positive clinical specimens, product trace-back and virologic testing and sequencing of HAV RNA from the product. Additionally, an online survey open to all Hawaii residents was conducted to estimate baseline commercial food consumption. We identified 292 confirmed HAV cases, of whom 11 (4%) were possible secondary cases. Seventy-four (25%) were hospitalised and there were two deaths. Among all cases, 94% reported eating at Oahu or Kauai Island branches of Restaurant Chain A, with 86% of those cases reporting raw scallop consumption. In contrast, a food consumption survey conducted during the outbreak indicated 25% of Oahu residents patronised Restaurant Chain A in the 7 weeks before the survey. Product trace-back revealed a single distributor that supplied scallops imported from the Philippines to Restaurant Chain A. Recovery, amplification and sequence comparison of HAV recovered from scallops revealed viral sequences matching those from case-patients. Removal of product from implicated restaurants and vaccination of those potentially exposed led to the cessation of the outbreak. This outbreak further highlights the need for improved imported food safety.