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PubMedDate not supplied

The effect of high-pressure processing on infectivity of Cryptosporidium parvum oocysts recovered from experimentally exposed Eastern oysters (Crassostrea virginica).

Shellfish have been identified as a potential source of Cryptosporidium infection for humans. The inactivation of Cryptosporidium parvum and other pathogens in raw molluscan shellfish would provide increased food safety for normal and at-risk consumers. The present study identified the efficacy of a non-thermal alternative food-processing treatment, high hydrostatic pressure processing (HPP), on the viability of C. parvum oocysts in the Eastern oysters Crassostrea virginica. Oysters were artificially exposed to 2 x 10(7) oocysts of the Beltsville strain of C. parvum in seawater and subjected to HPP treatments. The effects of the treatments were evaluated by inoculation of the processed oyster tissues into neonatal mice. High-pressure processing of shucked Eastern oysters at all pressures tested (305, 370, 400, 480, and 550 MPa) was significantly effective (P<0.05) in reducing the numbers of positive mouse pups fed treated oyster tissues exposed to C. parvum oocysts. A dose of 550 MPa at 180 s (s) of holding time produced the maximum decrease in numbers of C. parvum positive mouse pups (93.3%). Measurement of tristimulus color values of HPP-treated raw oysters at extended processing times from 120 s to 360 s at 550 MPa showed a small increase in whiteness of oyster meat. This non-thermal processing treatment shows promise for commercial applications to improve safety of seafood and reduce public health risks from cryptosporidiosis.

Animals↗

PubMed2006

Validation of a green fluorescent protein-labeled strain of Vibrio vulnificus for use in the evaluation of postharvest strategies for handling of raw oysters.

In this paper we describe a biological indicator which can be used to study the behavior of Vibrio vulnificus, an important molluscan shellfish-associated human pathogen. A V. vulnificus ATCC 27562 derivative that expresses green fluorescent protein (GFP) and kanamycin resistance was constructed using conjugation. Strain validation was performed by comparing the GFP-expressing strain (Vv-GFP) and the wild-type strain (Vv-WT) with respect to growth characteristics, heat tolerance (45 degrees C), freeze-thaw tolerance (-20(o) and -80 degrees C), acid tolerance (pH 5.0, 4.0, and 3.5), cold storage tolerance (5 degrees C), cold adaptation (15 degrees C), and response to starvation. Levels of recovery were evaluated using nonselective medium (tryptic soy agar containing 2% NaCl) with and without sodium pyruvate. The indicator strain was subsequently used to evaluate the survival of V. vulnificus in oysters exposed to organic acids (citric and acetic acids) and various cooling regimens. In most cases, Vv-GFP was comparable to Vv-WT with respect to growth and survival upon exposure to various biological stressors; when differences between the GFP-expressing and parent strains occurred, they usually disappeared when sodium pyruvate was added to media. When V. vulnificus was inoculated into shellstock oysters, the counts dropped 2 log(10) after 11 to 12 days of refrigerated storage, regardless of the way in which the oysters were initially cooled. Steeper population declines after 12 days of refrigerated storage were observed for both iced and refrigerated products than for slowly cooled product and product held under conservative harvest conditions. By the end of the refrigeration storage study (22 days), the counts of Vv-GFP in iced and refrigerated oysters had reached the limit of detection (10(2) CFU/oyster), but slowly cooled oysters and oysters stored under conservative harvest conditions still contained approximately 10(3) and >10(4) CFU V. vulnificus/oyster by day 22, respectively. The Vv-GFP levels in the oyster meat remained stable for up to 24 h when the meat was exposed to acidic conditions at various pH values. Ease of detection and comparability to the wild-type parent make Vv-GFP a good candidate for use in studying the behavior of V. vulnificus upon exposure to sublethal stressors that might be encountered during postharvest handling of molluscan shellfish.

Animals↗

PubMed1978

Incidence of Vibrio parahaemolyticus bacteriophages and other Vibrio bacteriophages in marine samples.

Vibrio bacteriophages were isolated by enrichment from 177 of 643 samples of marine molluscan shellfish, crustaceans, seawater, and sediments. The predominant bacteriophage types isolated were specific for some strains of Vibrio parahaemolyticus. A high frequency of phage isolations was also observed with strains of agar-digesting vibrios (21 of 56) and psychrophilic vibrios (14 of 72) that were originally isolated from non-shellfish growing areas. No bacteriophages were isolated against V. alginolyticus and only rarely for V. anguillarum even though these were the two most abundant species found in near-shore environments. No V. cholerae phages were isolated. It was also determined from quantitative studies on the Pacific oyster (Crassostrea gigas) obtained from two environments in Washington and Oregon that the titers of V. parahaemolyticus bacteriophages increased with increasing seasonal water temperatures and that this was proportional to the increase in numbers of mesophilic vibrios and not with the incidence of V. parahaemolyticus. Titers of V. parahaemolyticus bacteriophages occasionally exceeded 10(6) per g of oyster during the summer months. Specific V. parahaemolyticus bacteriophages were also isolated from market seafoods and other marine samples that originated in cold environments where no mesophilic vibrios are expected to be found. The possibility that V. parahaemolyticus bacteriophages originate from Vibrio spp. other than V. parahaemolyticus and the role of these bacteriophages in the ecology of marine vibrios are discussed.

Animals↗

PubMed1994

Detection of hepatitis A virus in environmental samples by antigen-capture PCR.

The efficacy of the antigen-capture PCR (AC-PCR) method for the detection of hepatitis A virus (HAV) in environmental samples was demonstrated. HAV was captured from a seeded liquid waste or a shellfish sample with homologous antibody and then heat denatured and subjected to reverse transcription and the PCR, all in the same tube. Subsequently, the AC-PCR products were analyzed by oligonucleotide probe hybridization in solution, agarose gel electrophoresis, and autoradiography. The AC-PCR detected as little as 0.053 PFU of cell culture-adapted HAV strain HM175/18f. The results of cDNA-RNA hybridization indicated that the particle/PFU ratio of this virus strain is approximately 79:1. Therefore, the detection limit of the AC-PCR was estimated to be four virus particles. No amplified products were observed when poliovirus 1, coxsackievirus A9, coxsackievirus B3, echovirus 6, reovirus 1, adenovirus type 40, human rotavirus type 1, and bovine enterovirus type 2 were tested, confirming the specificity of the assay. There were no differences between the nucleotide sequences of AC-PCR products of HAV strain HM175/18f and the sequences of wild-type HAV strain HM175 derived from molecularly cloned cDNA. Of 121 waste and shellfish samples tested by both plaque assays (PA) in cell cultures and the AC-PCR, 81 (67%) were positive and 31 (26%) were negative as determined by both methods, whereas 9 (7%) were positive as determined by the AC-PCR and negative as determined by the PA, and none were positive as determined by the PA and negative as determined by the AC-PCR.

Base Sequence↗

PubMed1995

Uptake and retention of Vibrio cholerae O1 in the Eastern oyster, Crassostrea virginica.

Vibrio cholerae 01, the causative agent of cholera, is known to persist in estuarine environments as endogenous microflora. The recent introduction of V. cholerae 01 into estuaries of the North and South American continents has stimulated the need to determine the effect of controlled purification on reducing this pathogen in edible molluscan shellfish. Experiments defined parameters for the uptake and retention of V. cholerae 01 in tissues of Crassostrea virginica, and these parameters were compared with those for Escherichia coli and Salmonella tallahassee, bacteria which are usually eliminated from moderately contaminated shellfish within 48 h. Oysters accumulated greater concentrations of V. cholerae 01 than E. coli and S. tallahassee. When V. cholerae 01 was exposed to controlled purification at 15, 19 and 25 degrees C over 48 h, it persisted in oysters at markedly higher levels than E. coli and S. tallahassee. The concentration of a V. cholerae 01-specific agglutinin did not positively correlate with the uptake or retention of V. cholerae 01. These data show that state and federally approved controlled purification techniques are not effective at reducing V. cholerae 01 in oysters.

Agglutinins↗

PubMed1996

Detection and analysis of a small round-structured virus strain in oysters implicated in an outbreak of acute gastroenteritis.

Outbreaks of shellfish-transmitted viral disease occur periodically, but frequently the causative agent is not identified. In November 1993, during investigation of a multistate outbreak of acute gastroenteritis, incriminated lots of oysters were collected. Oyster tissues (stomachs and digestive diverticula) were processed for virus extraction and nucleic acid purification. Human calicivirus sequences were sought by reverse transcriptase PCR using different primer sets. Amplicons were obtained from 9 of 10 shellfish samples from four different lots when primers specific for the outbreak virus strain were used. The specificity of the amplification was confirmed by hybridization. The amplicons from the nine positive oysters were cloned and sequenced. The sequence of each of the clones was identical to the others but showed some variation (7 of 81 bp) from the sequences obtained from the stools of three persons made III by the outbreak.

Animals↗

PubMed1998

Occurrence of Vibrio vulnificus biotypes in Danish marine environments.

During the unusually warm summer in Denmark in 1994, 11 clinical cases of Vibrio vulnificus infection were reported. These reports initiated an investigation of the occurrence of V. vulnificus biotypes in Danish marine environments. Samples of coastal water, sediment, shellfish, and wild fish were analyzed by preenrichment in alkaline peptone water amended with polymyxin B (2.0 x 10(4) U/liter) followed by streaking onto modified cellobiose-polymyxin B-colistin agar. V. vulnificus-like colonies were tested with a V. vulnificus-specific DNA probe. Low densities of V. vulnificus were detected in water (0.8 to 19 CFU/liter) from June until mid-September and in sediment (0.04 to > 11 CFU/g) from July until mid-November. The presence of V. vulnificus was strongly correlated with water temperature. However, we isolated V. vulnificus from water from a mussel farm at a lower temperature than previously reported (7 degrees C). In 1 of the 13 locations studied, V. vulnificus was found in mussels in 7 of 17 samples analyzed; this is the first report of V. vulnificus in European shellfish. V. vulnificus was also isolated from gills, intestinal contents, and mucus from wild fish. Although biotyping of 706 V. vulnificus strains isolated during our investigations revealed that the majority of the strains (99.6%) belonged to biotype 1, biotype 2 was detected in seawater at a low frequency (0.4%). Our findings provide further evidence that seawater can serve as a reservoir and might facilitate spread of V. vulnificus biotype 2 to eels, with subsequent spread to persons handling eels. In conclusion, our data demonstrate that V. vulnificus is ubiquitous in a temperature marine environment and that V. vulnificus biotype 2 is not strictly confined to eels.

Animals↗

PubMed1998

Reverse transcriptase PCR detection of astrovirus, hepatitis A virus, and poliovirus in experimentally contaminated mussels: comparison of several extraction and concentration methods.

Four methods of extraction and three methods of concentration of three enteric viruses from mussels were comparatively evaluated by reverse transcriptase PCR (RT-PCR). Shellfish were experimentally contaminated by immersion in seawater seeded with astrovirus, hepatitis A virus, or poliovirus. Sixty-gram samples of mussel tissues were processed by using borate buffer, glycine solution, saline beef, and saline beef-Freon extraction methods. The viruses were concentrated by precipitation with polyethylene glycol 6000 (PEG 6000) or PEG 8000 or by organic flocculation. RT-PCR was performed with RNA extracts from crude shellfish extracts and concentrates with and without Sephadex LH20 filtration. The glycine solution and borate buffer extraction methods resulted in significantly more RT-PCR-positive samples than the saline beef extraction method. We assessed the efficiency of 20 combinations of extraction and concentration methods. The borate buffer-organic flocculation, borate buffer-PEG 6000, and glycine solution-PEG 6000 combinations gave RT-PCR-positive results for all 27 samples analyzed for the three viruses. Detoxification of the samples by Sephadex LH20 filtration significantly decreased the efficiency of RT-PCR virus detection.

Animals↗

PubMed2006

Human and animal enteric caliciviruses in oysters from different coastal regions of the United States.

Food-borne diseases are a major cause of morbidity and hospitalization worldwide. Enteric caliciviruses are capable of persisting in the environment and in the tissues of shellfish. Human noroviruses (HuNoVs) have been implicated in outbreaks linked to shellfish consumption. The genetic and antigenic relatedness between human and animal enteric caliciviruses suggests that interspecies transmission may occur. To determine the occurrence of human and animal enteric caliciviruses in United States market oysters, we surveyed regional markets. Oysters were collected from 45 bays along the United States coast during the summer and winter of 2002 and 2003. Samples were analyzed by reverse transcription-PCR, and results were confirmed by hybridization and sequence analysis. Nine samples (20%) were positive for HuNoV genogroup II after hybridization. Animal enteric caliciviruses were detected in 10 samples (22%). Seven of these samples were positive for porcine norovirus genogroup II, and one sample was positive for porcine sapovirus after hybridization and confirmation by sequencing. Bovine noroviruses were detected in two samples, and these results were confirmed by sequencing. Five HuNoV samples sequenced in the polymerase region were similar to the norovirus genogroup II US 95/96 subset (genogroup II-4) previously implicated in diarrhea outbreaks. Different seasonal and state distributions were detected. The presence of animal enteric caliciviruses was associated with states with high livestock production. Although the presence of human caliciviruses in raw oysters represents a potential risk for gastroenteritis, disease confirmation by investigation of outbreaks is required. The simultaneous detection of human and animal enteric caliciviruses raises concerns about human infection or coinfection with human and animal strains that could result in genomic recombination and the emergence of new strains.

Animals↗

PubMed1966

Microbial flora of irradiated Dungeness crabmeat and Pacific oysters.

The microorganisms in Dungeness crabmeat (Cancer magister) and Pacific oysters (Crassostrea gigas) were identified by the replica-plating and computer analysis method. The initial flora of the shellfish and the flora change during storage at 7 C were determined. The microbial flora shifts in both shellfish were also determined after irradiation at 0.1 and 0.4 Mrad and during subsequent storage at 7 C. The Achromobacter species predominated in the initial flora of crabmeat (77.0%). The predominant position of this group increased to 99.2% after 0.1 Mrad and 100% after 0.4 Mrad. A large percentage of Lactobacillus was detected in oysters (55.0%). The Lactobacillus species were the predominant survivors after 0.1 Mrad (92.4%) but the predominant survivors after 0.4 Mrad were Achromobacter species (99.3%).

Alcaligenes↗

PubMed2001

Public health response to an incident of secondary chemical contamination at a beach in the United Kingdom.

OBJECTIVES: To gather enough data from a large scale investigation involving two health authorities, to assess the possible concentrations and routes of exposure and the consequent health implications. To use the data to decide whether a polluted beach should remain open to the public. In Spring 1997, a chemical incident came to light at a beach on the south coast of England when a local resident reported a sulphurous smell, visible signs of oil, and reduced numbers of fishing bait. The beach was situated adjacent to a former gasworks site and was accessible to the public. The incident was reported to the local authority and was initially investigated by the Environment Agency and the local authority. An Environment Agency report confirmed contamination of the beach with cyanide, ammonia, and polycyclic aromatic hydrocarbons (PAHs) with associated potential health risks. The incident was then referred to the local health authorities for investigation. METHODS: The investigation was carried out in four stages: comprehensive sampling and analysis to identify the extent of contamination, followed by an assessment of risk to health; establishment of a long term monitoring programme to identify any changes in contaminant concentrations; investigation of the effects of the contamination on shellfish; and review of the routine monitoring data and current sampling strategy. RESULTS: The initial investigation confirmed that the beach was contaminated, with the most likely source being the adjacent former gasworks site. The level of contamination was not found to be likely to pose a hazard to users of the beach. However, subsequent investigation of shellfish in the area led to warning signs being erected on the beach to prevent human consumption of mussels contaminated with PAHs. CONCLUSIONS: Several lessons can be learnt from this investigation, which can be applied to incident management more generally: the importance of collaboration and coordination; the need for early involvement of the health authority; and the importance of carrying out appropriate sampling and analysis as soon as possible, to assess the risk to health and the environment.

Ammonia↗

PubMed1978

Steric hindrance as a factor in the reaction of labeled antibody with cell surface antigenic determinants.

The binding of rabbit anti-human IgG labeled with 125I, shellfish glycogen or ferritin to human IgG attached to the surface of rabbit RBC with chromic chloride was studied. Maximum binding was noted with 125I labeled antibody. Slightly but consistently less binding was found with shellfish glycogen labeled antibody. The binding of ferritin labeled antibody was strikingly reduced--usually one-third or less of that found with 125I labeled antibody alone. This suggests that under the conditions of these experiments, the attachment of large labels to antibody molecules results in reduced antibody binding to surface antigen. Steric hindrance is probably at least in part responsible for this reduced binding.

Animals↗

PubMed2004

Norovirus foodborne outbreaks associated with the consumption of oysters from the Etang de Thau, France, December 2002.

In January 2003, the Institut de Veille Sanitaire received notification of clusters of gastroenteritis (GE) thought to be associated with consumption of oysters harvested from Etang de Thau in the south of France. At the same time Italy reported an outbreak (200+ cases) associated with oysters from the Etang de Thau. An investigation was carried out to determine the source and vehicle of the outbreaks. Descriptive analysis of reported clusters in France, microbiological analysis of stool and oyster samples, genotyping of noroviruses and an environmental investigation of the Etang de Thau were carried out. A retrospective cohort study was also undertaken among those attending a number of family meals in Paris. Thirteen family clusters in four districts of France (69 cases) could be attributed to the consumption of Thau oysters based on descriptive evidence. Oysters distributed at an office in Paris and consumed at fourteen family meals between 19 and 24 December led to a further outbreak. In this outbreak the attack rate was 21/36 (58%) for Thau oyster consumers and 0/22 for non-consumers (p=0.00002). Noroviruses (genogroups I and II) were found in stool samples from four clusters and oysters from three clusters (including Paris). Environmental investigations revealed heavy rainfall, an overflow of a water purification station and faecal contamination of the Etang de Thau in December. Oysters from the Etang de Thau were responsible for a number of clusters of norovirus GE in winter 2002 in France and also in Italy. High Escherichia Coli levels in Thau water and shellfish led to an official request, mid-December, for oyster purification before distribution. This was not possible, due to lack of purification facilities. This investigation has contributed to a change in the way that shellfish harvesting areas are classified in France.

Adolescent↗

PubMed2000

Prevalence of high-risk food consumption and food-handling practices among adults: a multistate survey, 1996 to 1997. The Foodnet Working Group.

Risk factors for foodborne diseases include consumption of high-risk foods and unsanitary food-handling practices; however, little is known about the prevalence of these risk factors in the general population. A survey was done in five FoodNet sites (California, Connecticut, Georgia, Minnesota, Oregon) to determine the prevalence of these risk factors in the population. A total of 7,493 adults were interviewed by telephone between 1 July 1996 and 30 June 1997. Results showed that 1.5% drank raw milk, 1.9% ate raw shellfish, 18% ate runny egg, 30% preferred pink hamburger, 93% said they almost always washed their cutting board after cutting raw chicken, and 93% said they almost always washed their hands after handling raw meat or poultry, during 5 days before interview. The results differed by state and demographic group. Consumption of raw shellfish (3.2%) and undercooked hamburger (43%) were more common in Connecticut than other states. Raw milk consumption was more common among people who lived on a farm (8.6%) compared with people who lived in a city or urban area (1.1%). Preference for undercooked hamburger was more common among men (35%), young adults (18 to 25 years, 33%), people with college education (38%), and among people with household income of more than $100,000/year (49%). African-Americans were less likely to prefer undercooked hamburger compared to other racial groups (10% versus 30%). Young adults compared to older adults were less likely to wash their hands after handling raw chicken (88% versus 95%), and men washed their hands less often than women (89% versus 97%). Although there were statistical differences between demographic groups, they are insufficient to warrant targeted educational programs.

Adult↗

PubMed2003

Persistence of hepatitis A virus in oysters.

We investigated the ability of hepatitis A virus (HAV) to persist for up to 6 weeks in Eastern oysters (Crassostrea virginica). Viral RNA was detected by reverse transcription-polymerase chain reaction 6 weeks after 16 h of exposure to 90,000 PFU (180 PFU/ml of seawater) of HAV. Assaying for infectious virus in oysters that received a daily feeding of phytoplankton recovered 3,800, 650, and 500 PFU of HAV 1, 2, and 3 weeks after contamination with 90,000 PFU of HAV, respectively. However, no infectious HAV was isolated from oysters 4, 5, or 6 weeks after contamination. These results support the position that shellfish depuration is insufficient for the complete removal of infectious viruses. Extended relay times (in excess of 4 weeks) may be required to produce virologically safe shellfish.

Animals↗

PubMed2006

Cooking mussels (Mytilus galloprovincialis) by steam does not destroy the infectivity of Cryptosporidium parvum.

The consumption of shellfish has increased considerably worldwide, with an associated increase in foodborne illnesses. Among the bivalves, the mussels are usually cooked by steam, which constitutes a typical dish in several regions. In this article, we demonstrate that this preparation is not sufficient to destroy completely the infectivity of Cryptosporidium parvum. Oocysts recovered from experimentally contaminated mussels (Mytilus galloprovincialis) were infectious to neonatal mice after cooking. Although, to date, no official cases of cryptosporidiosis linked to shellfish consumption have been reported, we recommend that people with reduced immunity avoid this type of food because they are at high risk of being infected with Cryptosporidium spp. after eating raw or undercooked contaminated bivalves.

Animals↗

PubMed2006

Phenotypic and genotypic characterization of Salmonella enterica serotype paratyphi B isolates from environmental and human sources in Galicia, Spain.

Salmonella serotype Paratyphi B isolates obtained from shellfish and human infections in Galicia (northwest Spain) from 1998 were investigated by different phenotypic and genetic methods to evaluate their systemic or enteric nature. Isolates were investigated for D-tartrate fermentation, presence of genes encoding the effector proteins sopE1 and avrA, pulsed-field gel electrophoresis profile, and antimicrobial susceptibility. Systemic variant strains (dT-) were the dominant among the marine environment isolates. All dT- isolates were sopE1 positive and avrA negative, presented an indistinguishable electrophoresis profile, and were grouped in a single cluster. More electrophoresis heterogeneity was observed among dT+ isolates. Only two isolates showed resistance to any of the 16 antibiotics included in our panel. The present study identified the marine environment as a potential natural source of systemic variant isolates of Salmonella Paratyphi B. The presence of systemic variant isolates of Salmonella Paratyphi B in the marine environment is of notable public health significance as a result of the potential risk of acquiring enteric fever linked to the consumption of raw shellfish.

Animals↗

PubMed1986

Plesiomonas enteric infections in the United States.

Thirty-one persons nationwide from whom Plesiomonas shigelloides was isolated in large numbers from stool in 1984 were compared with 62 matched control subjects. Infection with P. shigelloides was strongly associated with eating uncooked shellfish, usually raw oysters, in the 48 hours before the onset of illness (p less than 0.00001) and with foreign travel (p less than 0.00006), usually to Mexico. Most ill persons had self-limited diarrhea with blood and mucus in stool and other clinical findings that suggested enteroinvasiveness of infecting organisms. Two patients developed their illnesses after taking ampicillin for reasons unrelated to diarrhea; plesiomonads recovered from their stools were resistant to ampicillin. Seven persons with gastrointestinal complaints had alleviation or resolution of their symptoms after taking antimicrobial agents to which recovered plesiomonads were susceptible. These findings suggest that P. shigelloides may cause enteric disease in the normal host, that it may be acquired from eating uncooked shellfish, and that it may be a cause of travelers' diarrhea.

Adolescent↗