Effect of high hydrostatic pressure on four genotypes of F-specific RNA bacteriophages.

AIMS: The pressure responses of four genotypes of F-specific RNA bacteriophages, f2, GA, Qbeta and SP, were evaluated with respect to pressure magnitude, treatment temperature and suspending medium. METHOD AND RESULTS: The pressure responses were studied with respect to pressure magnitude (350 to 600 MPa), treatment temperature (-10 to 50 degrees C) and suspending media. Phages f2 and GA had much higher pressure resistances than Qbeta and SP. Pressure resistances of Qbeta and SP were enhanced with increase in salt concentrations in the range of 350 to 600 MPa from -10 to 50 degrees C in PBS. Qbeta and SP had greater pressure resistances when suspended in phosphate-buffered saline (PBS) with added glucose (5%, w/w), UHT whole milk and Dulbecco's Modified Eagle's Medium plus 10% fetal bovine sera than they did in PBS. Two surfactants, sucrose laurate and monolaurin, and one chelating agent, ethylenediamine tetraacetic acid (EDTA), increased the pressure resistance of Qbeta and SP, but had modest effect on either f2 or GA. CONCLUSIONS: Four representative F-specific RNA bacteriophages, f2 (serotype I), GA (serotype II), Qbeta (serotype III) and SP (serotype IV) showed different resistances to hydrostatic pressure in the range of 350-600 MPa. SIGNIFICANCE AND IMPACT OF THE STUDY: This study screened for practical surrogates of HAV for validation of commercial high hydrostatic pressure processing.

Response of four types of coliphages to high hydrostatic pressure.

Pressure inactivation of four types of coliphages, varphiX 174 (ssDNA virus), MS2 (ssRNA virus), lambda imm434 (dsDNA virus) and T4 (dsDNA virus), was studied to evaluate their potential as human enteric viral surrogates for use in validation of commercial pressure processing treatments. Phage varphiX 174 demonstrated an unexpected high resistance to pressure with no more than 1-log(10) reduction observed following exposures to 350-600 MPa. There was no greater than 1-log(10) reduction below 500 MPa for MS2 in modified phosphate-buffered saline, but a 3.3-log(10) reduction was observed for MS2 pressurized at 600 MPa. Coliphages lambda imm434 and T4 were relatively sensitive to pressure in demonstrating inactivation at 350 MPa. At 21 degrees C, lambda imm434 was inactivated in modified phosphate-buffered saline or Dulbecco's Modified Eagle's Medium plus 5% fetal bovine sera by at least 7.5-log(10) when exposed to 400 MPa for 5 min. Treatment at 450 MPa for 5 min was necessary to obtain a log(10) reduction of 6-7 for T4.

Selective accumulation may account for shellfish-associated viral illness.

From 1991 through 1998, 1,266 cases of shellfish-related illnesses were attributed to Norwalk-like viruses. Seventy-eight percent of these illnesses occurred following consumption of oysters harvested from the Gulf Coast during the months of November through January. This study investigated the ability of eastern oysters (Crassostrea virginica) to accumulate indicator microorganisms (i.e., fecal coliforms, Escherichia coli, Clostridium perfringens, and F(+) coliphage) from estuarine water. One-week trials over a 1-year period were used to determine if these indicator organisms could provide insight into the seasonal occurrence of these gastrointestinal illnesses. The results demonstrate that oysters preferentially accumulated F(+) coliphage, an enteric viral surrogate, to their greatest levels from late November through January, with a concentration factor of up to 99-fold. However, similar increases in accumulation of the other indicator microorganisms were not observed. These findings suggest that the seasonal occurrence of shellfish-related illnesses by enteric viruses is, in part, the result of seasonal physiological changes undergone by the oysters that affect their ability to accumulate viral particles from estuarine waters.

A method to detect low levels of enteric viruses in contaminated oysters.

Direct isolation and identification of pathogenic viruses from oysters implicated in gastroenteritis outbreaks are hampered by inefficient methods for recovering viruses, naturally occurring PCR inhibitors, and low levels of virus contamination. In this study we focused on developing rapid and efficient oyster-processing procedures that can be used for sensitive PCR detection of viruses in raw oysters. Poliovirus type 3 (PV3) Sabin strain was used to evaluate the efficacy of virus recovery and the removal of PCR inhibitors during oyster-processing procedures. These procedures included elution, polyethylene glycol precipitation, solvent extraction, and RNA extraction. Acid adsorption-elution in which glycine buffer (pH 7.5) was used was found to retain fewer inhibitors than direct elution in which glycine buffer (pH 9.5) was used. RNA extraction in which a silica gel membrane was used was more effective than single-step RNA precipitation for removing additional nonspecific PCR inhibitors. The final 10-microl volume of RNA concentrates obtained from 2 g of oyster tissue (concentration factor, 200-fold) was satisfactory for efficient reverse transcription-PCR detection of virus. The overall detection sensitivity of our method was 1 PFU/g of oyster tissue initially seeded with PV3. The method was utilized to investigate a 1998 gastroenteritis outbreak in California in which contaminated oysters were the suspected disease transmission vehicle. A genogroup II Norwalk-like virus was found in two of three recalled oyster samples linked by tags to the harvest dates and areas associated with the majority of cases. The method described here improves the response to outbreaks and can be used for rapid and sensitive detection of viral agents in outbreak-implicated oysters.

Occurrence of male-specific bacteriophage in feral and domestic animal wastes, human feces, and human-associated wastewaters.

Male-specific bacteriophage (MSB) densities were determined in animal and human fecal wastes to assess their potential impact on aquatic environments. Fecal samples (1,031) from cattle, chickens, dairy cows, dogs, ducks, geese, goats, hogs, horses, seagulls, sheep, and humans as well as 64 sewerage samples were examined for MSB. All animal species were found to harbor MSB, although the great majority excreted these viruses at very low levels. The results from this study demonstrate that in areas affected by both human and animal wastes, wastewater treatment plants are the principal contributors of MSB to fresh, estuarine, and marine waters.