Miles L. Motes

Molecular confirmation of oysters as the vector for hepatitis a in a 2005 multistate outbreak

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.

Incidence of Listeria spp. in shrimp, oysters, and estuarine waters.

A total of 227 samples, including oysters, shrimp, and water, was collected along the U.S. Gulf Coast and examined to determine the presence of Listeria spp. Listeria spp. were recovered more frequently from shrimp than from water but were not recovered from oysters. Recovery of Listeria spp. from shrimp and waters was improved at temperatures ≤20°C; however, recovery was not affected by salinity or related to the fecal coliform standard for shellfish-growing waters. Although only 5% of the test samples were positive for L. monocytogenes , all Listeria positive shrimp contained L. monocytogenes . The incidence of Listeria spp. in shrimp was low; nevertheless, shrimp represent a potential source of Listeria contamination to processing plants and their products.

Evaluation of an alkaline phosphatase-labeled DNA probe for enumeration of Vibrio vulnificus in Gulf Coast oysters

Abstract A direct plating method and an FDA method were compared for enumeration of Vibrio vulnificus in Gulf Coast oysters. The direct plating method was based on hybridization of colony lifts and used an alkaline phosphatase-labeled oligonucleotide probe that targeted the cytolysin gene (Direct-VVAP). The FDA method employs most probable number analysis with confirmation of suspect isolates by enzyme immunoassay (MPN-EIA). Indigenous V . vulnificus levels in oysters harvested from Florida, Alabama and Louisiana throughout the year and in Gulf Coast market oysters ranged from 5 g −1 . Similar V. vulnificus levels ( r =0.66) were found with these two procedures in freshly harvested oysters collected between April and October. Measurement variance was much greater, however, with the MPN procedure (0.118) than with the DNA probe procedure (0.004). In market oysters, the MPN-EIA procedure gave 0.4 log 10 higher estimates than the Direct-VVAP method, but the methods were closely correlated ( r =0.83). Confirmation procedures were in agreement >90% of the time, as suspect isolates confirmed by one procedure were identified by the other procedure. Except when V . vulnificus densities are low ( −1 ), the Direct-VVAP method provides an alternative procedure that is more rapid and precise than the MPN-EIA analysis.

Use of Molecular Epidemiology to Confirm a Multistate Outbreak of Hepatitis a Caused by Consumption of Oysters

The 39 oyster consumption-related cases of hepatitis A reported in 2005 represent the first large outbreak of hepatitis A associated with shellfish consumption in the United States in >15 years. This is the first outbreak investigation in which an identical hepatitis A virus sequence was obtained from both the implicated food product and case patients.

Recovery of Heat-Stressed Listeria monocytogenes from Experimentally and Naturally Contaminated Shrimp

A method was developed to enhance recovery of thermally stressed Listeria monocytogenes from internally contaminated shrimp. Shrimp tail meat was inoculated with 105 L. monocytogenes cells/g and boiled for 1-5 min. Thermally stressed L. monocytogenes cells were recovered following cold enrichment for 3 d without broth. Methods of the Food and Drug Administration and the U.S. Department of Agriculture for isolating L. monocytogenes permitted recovery of the organism from shrimp boiled for 1 min: however, with the new method, L. monocytogenes cells were recovered from shrimp boiled up to 5 min. No Listeria spp. were recovered from naturally contaminated, frozen, imported shrimp after 1 min of boiling.

Effects of a commercial heat-shock process on Vibrio vulnificus in the American oyster, Crassostrea virginica, harvested from the Gulf Coast.

Oysters (Crassostrea virginica) harvested from the Gulf Coast, containing 10(2) to 10(4) most probable number (MPN) per gram of Vibrio vulnificus, were subjected to a commercial heat-shock process. After 1 to 4 min at internal oyster meat temperatures exceeding 50 degrees C, shellstock oysters were shucked, chilled, washed, and packed. V. vulnificus and total bacterial levels in Gulf Coast oysters were significantly reduced from 1 to 4 logs in the finished product. Similar reductions were not observed in shellstock oysters that were subject to conventional processing. Under the National Shellfish Sanitation Program, heat shocking is an acceptable process to use to assist in the shucking of shellstock. This research revealed that the heat-shock process may also serve to significantly reduce V. vulnificus in summer Gulf Coast oysters.

Occurrence of Toxigenic Vibrio cholerae O1 in Oysters in Mobile Bay, Alabama: An Ecological Investigation

Toxigenic Vibrio cholerae O1 Inaba, resembling the epidemic Latin American strains (C6706, C6707), was recovered from oysters taken from Mobile Bay, Alabama, on five separate occasions between July 1991 and September 1992. Levels of toxigenic V. cholerae in the oysters, estimated by the most probable number procedure, ranged from 101 to 107 per 100 g. Isolates characterized by pulsed field gel electrophoresis resembled isolates previously recovered from five cargo ships docked at Gulf of Mexico ports. This study details the first reported isolation of toxigenic V. cholerae O1 from oysters in U.S. coastal waters. As with the Gulf Coast strain, the occurrence of the epidemic strain seems to be sporadic and essentially limited to the warmer months.

Field evaluation of the MUG assay for enumerating Escherichia coli in seawater and oysters from southeastern United States

Oysters and seawater collected from the southeastern United States were examined for fecal coliforms and Escherichia coli , using the current procedure of the American Public Health Association (APHA) and the fluorogenic 4-methylumbelliferyl-β-D-glucuronide (MUG) modified APHA procedure. After the presence of E. coli in both methods was confirmed by conventional IMViC procedures, there was no significant difference between method means at the α = 0.05 level. In oysters, low confirmation rates of 67 and 77% were observed by the APHA and the MUG methods, respectively. Seawater had the greatest confirmation rates (95%) by the MUG method. The MUG method may be a suitable alternative to the current APHA method for the microbiological evaluation of oysters and seawater.

Comparison of Three International Methods with APHA Method for Enumeration of Escherichia coli in Estuarine Waters and Shellfish

Three international methods were evaluated for enumerating Escherichia coli in estuarine waters, oysters ( Crassostrea virginica ), mussels ( Mytilus edilus ) and clams ( Mercenaria mercenaria ). Results of the French most probable number (MPN) method, a modification of the MacKenzie, Taylor and Gilbert (1948) method, were obtained within 48 h and compared favorably with those obtained by the standard American Public Health Association (APHA) MPN procedure in all sample types. Results of the Australian Anderson and Baird-Parker plate count method, obtained within 24 h, were significantly lower than those obtained with the standard APHA procedure for all sample types. Results of the British roll tube method, a 24-h direct count method, compared favorably with the standard APHA procedure only for mussels and waters.