Randomized, double-blinded clinical trial for human norovirus inactivation in oysters by high hydrostatic pressure processing.

Contamination of oysters with human noroviruses (HuNoV) constitutes a human health risk and may lead to severe economic losses in the shellfish industry. There is a need to identify a technology that can inactivate HuNoV in oysters. In this study, we conducted a randomized, double-blinded clinical trial to assess the effect of high hydrostatic pressure processing (HPP) on Norwalk virus (HuNoV genogroup I.1) inactivation in virus-seeded oysters ingested by subjects. Forty-four healthy, positive-secretor adults were divided into three study phases. Subjects in each phase were randomized into control and intervention groups. Subjects received Norwalk virus (8FIIb, 1.0 × 10(4) genomic equivalent copies) in artificially seeded oysters with or without HPP treatment (400 MPa at 25°C, 600 MPa at 6°C, or 400 MPa at 6°C for 5 min). HPP at 600 MPa, but not 400 MPa (at 6° or 25°C), completely inactivated HuNoV in seeded oysters and resulted in no HuNoV infection among these subjects, as determined by reverse transcription-PCR detection of HuNoV RNA in subjects' stool or vomitus samples. Interestingly, a white blood cell (granulocyte) shift was identified in 92% of the infected subjects and was significantly associated with infection (P = 0.0014). In summary, these data suggest that HPP is effective at inactivating HuNoV in contaminated whole oysters and suggest a potential intervention to inactivate infectious HuNoV in oysters for the commercial shellfish industry.

Effect of high hydrostatic pressure processing on freely suspended and bivalve-associated T7 bacteriophage.

The effectiveness of hydrostatic pressure processing (HPP) for inactivating viruses has been evaluated in only a limited number of studies, and most of the work has been performed with viruses freely suspended in distilled water. In this work, HPP inactivation of freely suspended and shellfish-associated bacteriophage T7 was studied. T7 was selected in hopes that it could serve as a model for animal virus behavior. Clams (Mercenaria mercenaria) and oysters (Crassostrea virginica) were homogeneously blended separately and inoculated with bacteriophage T7. The inoculated bivalve meat and the freely suspended virus samples were subjected to HPP under the following conditions: 2, 4, and 6 min at 241.3, 275.8, and 344.7 MPa pressure and temperatures of 29.4 to 35, 37.8 to 43.3, and 46.1 to 51.7 degrees C. Reductions of 7.8 log PFU (100% inactivation) were achieved for freely suspended T7 at 344.7 MPa for 2 min at 37.8 to 43.3 degrees C. At 46.1 to 51.7 degrees C, T7 associated with either clams or oysters was inactivated at nearly 100% (>4 log PFU) at all pressure levels and durations tested. These results indicate that T7 is readily inactivated by HPP under the proper conditions, may be made more susceptible to HPP by mixing with shellfish meat, and may serve as a viable model for the response of several animal viruses to HPP.

Effects of high pressure processing on Toxoplasma gondii oocysts on raspberries.

Oocysts are the environmentally resistant life stage of Toxoplasma gondii. Humans can become infected by accidentally ingesting the oocysts in water or from contaminated produce. Severe disease can occur in immunocompromised individuals, and nonimmune pregnant women can infect their offspring. Chronic infection is associated with decreased mental functions, vision and hearing problems, and some mental disorders such as schizophrenia. High pressure processing (HPP) is a commercial method used to treat food to eliminate pathogens. Treatment of produce to eliminate viable T. gondii oocysts would provide a means to protect consumers. The present study was done to better define the effects of HPP on oocysts placed on raspberries. Raspberries were chosen because they are a known source of a related human intestinal parasite, Cyclospora cayetanensis. Raspberries were inoculated with 5 x 10(4) oocysts of the VEG strain of T. gondii for 20 hr prior to HPP. Individual raspberries were exposed to 500 MPa, 400 MPa, 340 MPa, 300 MPa, 270 MPa, 250 MPa, 200 MPA, 100 MPa, or no MPa treatment for 60 sec in a commercial HPP unit (1 MPa = 10 atm = 147 psi). Treatment of raspberries with 340 MPa for 60 sec was needed to render oocysts spot inoculated on the raspberries noninfectious for mice. Treatment of raspberries with 200 MPa or less for 60 sec was not effective in rendering oocysts noninfectious for mice.

Effects of high hydrostatic pressure on embryonation of Ascaris suum eggs.

High hydrostatic pressure processing (HPP) has been shown to be an effective non-thermal means of inactivating microorganisms from various food products. Little information is available regarding the effects of HPP on metazoan parasites. Outbreaks of food-borne disease have been associated with importation of food contaminated with fecal material. Ascaris suum is used as a surrogate model metazoan parasite for the human roundworm, Ascaris lumbricoides, to study the effects of treatments on the inactivation of eggs in sludge. The present study was conducted to determine the effects of HPP on A. suum eggs. Unembryonated A. suum eggs were subjected to 138-552 megapascals (MPa) for 10-60s in a commercial HPP unit. Embryonation was induced after HPP treatments by incubating eggs in 0.01N sulfuric acid at room temperature. After 21 days, 100 eggs were examined per treatment using a light microscope and the percent of embryonated eggs was determined. Embryonation was induced in 38-76% eggs that were subjected to 138 and 270MPa. No embryonation was observed in eggs exposed to pressures of 241MPa or more for 60s or in eggs exposed to 276MPa for 10-30s. These results indicate that HPP treatment could be used to protect contaminated food items by inactivating A. suum eggs and may also have potential in reducing food-borne illness resulting from fecal contamination.

Effects of high-pressure processing on Toxoplasma gondii tissue cysts in ground pork.

Ingestion of Toxoplasma gondii tissue cysts can result in severe disease in immunocompromised individuals and pregnant women. Treatment of meat and meat products to eliminate viable T. gondii tissue cysts would provide a means to protect consumers. In this study, we examined the effects of high-pressure processing (HPP) on ground pork containing viable tissue cysts of the VEG strain of T. gondii. Ground pork containing tissue cysts was exposed to 400, 300, 200, 100, or 0 MPa treatment for 30, 60, or 90 sec in a commercial HPP unit. The HPP-treated ground pork was subjected to acid-pepsin digestion and bioassayed in mice. The results of the mouse bioassay revealed that none of the mice inoculated with tissue cysts exposed to 400 or 300 MPa became infected, whereas all mice inoculated with tissue cysts exposed to 200, 100, or 0 MPa became infected with T. gondii regardless of exposure time. Results indicate that HPP treatment of ground pork with 300 MPa of pressure will render tissue cysts of T. gondii nonviable and make pork safe for human consumption.

Effects of high-pressure processing on in vitro infectivity of Encephalitozoon cuniculi.

High-pressure processing (HPP) has been shown to be an effective means of eliminating bacteria and destructive enzymes from a variety of food products. HPP extends the shelf life of products while maintaining the sensory features of food and beverages. In this study, we examined the effects of HPP on the infectivity of Encephalitozoon cuniculi spores in vitro. Spores were exposed to between 140 and 550 MPa for 1 min in a commercial HPP unit. Following treatment, the spores were loaded onto cell culture flasks or were kept for examination by transmission electron microscopy. No effect was observed on the infectivity of spores treated with 140 MPa. Spores treated with between 200 and 275 MPa showed reduction in infectivity. Following treatment of 345 MPa or more, spores were unable to infect host cells. No morphologic changes were observed in pressure-treated spores with transmission electron microscopy.

Effects of high pressure processing on infectivity of Toxoplasma gondii oocysts for mice.

High pressure processing (HPP) has been shown to be an effective non-thermal method of eliminating non-spore forming bacteria from a variety of food products. The shelf-life of the products is extended and the sensory features of the food are not or only minimally effected by HPP The present study examined the effects of HPP using a commercial scale unit on the viability of Toxoplasma gondii oocysts. Oocysts were exposed from 100 to 550 MPa for 1 min in the HPP unit and then HPP treated oocysts were orally fed to groups of mice. Oocysts treated with 550 MPa or less did not develop structural alterations when viewed with light microscopy. Oocysts treated with 550 MPa, 480 MPa, 400 Mpa, or 340 MPa were rendered noninfectious for mice. Mice fed oocysts treated with no or 100 to 270 MPa became infected and most developed acute toxoplasmosis and were killed or died 7 to 10 days after infection. These results suggest that HPP technology may be useful in the removal of T. gondii oocysts from food products.

Survival of Toxoplasma gondii oocysts in Eastern oysters (Crassostrea virginica).

Toxoplasma gondii has recently been recognized to be widely prevalent in the marine environment. It has previously been determined that Eastern oysters (Crassostrea virginica) can remove sporulated T. gondii oocysts from seawater and that oocysts retain their infectivity for mice. This study examined the long-term survival of T. gondii oocysts in oysters and examined how efficient oysters were at removing oocysts from seawater. Oysters in 76-L aquaria (15 oysters per aquarium) were exposed to 1 x 10(6) oocysts for 24 hr and examined at intervals up to 85 days postexposure (PE). Ninety percent (9 of 10) of these oysters were positive on day 1 PE using mouse bioassay. Tissue cysts were observed in 1 of 2 mice fed tissue from oysters exposed 21 days previously. Toxoplasma gondii antibodies were found in 2 of 3 mice fed oysters that had been exposed 85 days previously. In another study, groups of 10 oysters in 76-L aquaria were exposed to 1 x 10(5), 5 x 10(4), or 1 x 10(4) sporulated T. gondii oocysts for 24 hr and then processed for bioassay in mice. All oysters exposed to 1 x 10(5) oocysts were infected, and 60% of oysters exposed to 5 x 10(4) oocysts were positive when fed to mice. The studies with exposure to 1 x 10(4) oocysts were repeated twice, and 10 and 25% of oysters were positive when fed to mice. These studies indicate that T. gondii can survive for several months in oysters and that oysters can readily remove T. gondii oocysts from seawater. Infected filter feeders may serve as a source of T. gondii for marine mammals and possibly humans.

Effects of high hydrostatic pressure processing on embryonation of Toxocara canis eggs.

Toxocara canis is a zoonotic nematode parasite that can be transmitted to humans by food or water contaminated with T. canis eggs from infected dog feces. High-pressure processing (HPP) is a useful alternative to thermal treatments to eliminate pathogens from foods. Most of the research on HPP has focused on prokaryotes, but little is known about its effects on eukaryotic organisms. We evaluated the ability of HPP to affect embryonation of T. canis eggs to test the hypothesis that HPP treatment can delay development of T. canis eggs. Efficacy of HPP was determined by using an embryonation assay on T. canis eggs from naturally infected puppies. For each treatment, 2500 T. canis eggs in tap water were placed in sealable plastic bags and subjected to 138-400 megapascals (MPa; 1 MPa=10 atm=147 psi) for 60 s in a commercial HPP unit. We found that treatment with 300 or 400 MPa for 60 s killed 100% of eggs using embryonation as the standard. Treatment with 250, 241, and 207 MPa was less effective and killed 80%, 56%, and 8% of eggs, respectively. Results from this study suggest that HPP may be a useful treatment to protect foods from T. canis contamination.

Effect of cooling rates and temperatures on quality and safety of quahog clams (Mercenaria mercenaria).

The model ordinance in the National Shellfish Sanitation Program's Guide for the Control of Molluscan Shellfish was initially established for oysters; however, the clam industry also follows the protocol. Rapid cooling during periods when the growing waters exceed 80 °F (26.7 °C) results in cold shock, which causes unacceptable mortalities in clams. The clam industry was looking for a procedure to lower the clams to the standard temperature while minimizing shell shock mortalities during the warm summer months. Three tempering treatments were examined, and total aerobic plate counts (APCs) and most-probable-number (MPN) counts of Vibrio, V. parahaemolyticus, and fecal coliforms were enumerated. In treatment 1 (control), clams were harvested, held for 5 h at 90 °F (32.2 °C), and then moved to 45 °F (7.2 °C) for storage. In treatment 2, clams were harvested and held for 5 h at 90 °F (32.2 °C), followed by 12 h at 65 °F (18.3 °C) and 12 h at 55 °F (12.8 °C), and then were moved to 45 °F (7.2 °C) for long-term storage. In treatment 3, clams were harvested and held for 5 h at 90 °F (32.2 °C), followed by 24 h at 55 °F (12.8 °C) before being moved to 45 °F (7.2 °C) for long-term storage. Three replicate trials were performed with triplicate analyses during late June through early to mid-August. The current National Shellfish Sanitation Program standard is treatment 1; it contained statistically (P ≤ 0.05) higher total APCs than treatments 2 and 3 throughout the 21-day storage period. APCs ranged from 2.3 × 10(4) immediately after harvest to 2.7 × 10(6), 1.6 × 10(5), and 4.8 × 10(5) for treatments 1, 2, and 3, respectively, after 14 days of storage. A statistical analysis showed that treatments 2 and 3 had significantly lower total MPN per gram Vibrio than treatment 1 on day 7 but were equal to treatment 1 on days 1 and 14. MPN per gram for V. parahaemolyticus was statistically lower in treatments 2 and 3 than in treatment 1 on storage days 1 and 7. However, on day 14, treatment 3 was significantly lower than treatments 1 and 2. There was no statistical difference for fecal coliforms. The greatest mortality occurred in treatment 1 (87.4%), followed by treatment 2 (83.3%) and treatment 3 (66.0%). The outcome of this research clearly shows that treatments 2 and 3 can cool clams to a temperature of 45 °F (7.2 °C) without compromising quality or safety and can reduce the number of dead clams introduced into the marketplace.