The fate of calicivirus in oysters in a 10-day depuration was assessed. The norovirus gene was persistently detected from artificially contaminated oysters during the depuration, whereas feline calicivirus in oysters was promptly eliminated. The prolonged observation of norovirus in oysters implies the existence of a selective retention mechanism for norovirus within oysters.Noroviruses have been one of the major causative agents of acute gastroenteritis and are often related to food poisoning from eating oysters (12,15). Cases of food poisoning caused by norovirus-contaminated raw oysters have been increasing, and marine industries related to oysters have been significantly damaged by the contamination of cultivated oysters with noroviruses. Many researchers are devoted to developing procedures for efficient removal or inactivation of pathogenic viruses in contaminated oysters (5,14), and viral surrogates have been employed to evaluate the removal or inactivation efficiency of noroviruses in oysters because there are no suitable cell culture systems to cultivate noroviruses (25). Feline calicivirus (FCV) is one of the most frequently used surrogates (1,6,11,26). Norovirus and FCV belong to the same family, Caliciviridae, which are small, nonenveloped, icosahedral viruses with a linear, positive-sense, single-stranded RNA genome (7). Since norovirus and FCV have almost the same configuration, these two viruses are expected to have similar fates in oysters. However, the similarity or discrepancy of behaviors between norovirus and FCV in oysters has not been significantly evaluated thus far.In this study, the persistency of norovirus genogroup II (GII) in oysters was compared with that of the FCV f4 strain. Oysters (Crassostrea gigas) were artificially contaminated with norovirus GII and FCV f4 for 72 h; the contaminated oysters were moved to a water body without viruses for a 10-day depuration. The gene concentrations of test viruses in oysters were quantified with the real-time quantitative reverse transcription-PCR (qRT-PCR) during the contamination and depuration periods. FCV strain f4 used in this study was given by Y. Tohya, University of Tokyo, and was multiplied in CRFK cells cultivated with minimum essential medium including 10% bovine serum according to Tohya et al. (23). Norovirus GII used in this study was purified from a stool specimen of a patient with acute infectious gastroenteritis in Miyagi Prefecture, Japan. About 20 g of stool was suspended in 200 ml of distilled water and centrifuged at 10,000 ϫ g for 30 min. The supernatant, including norovirus particles, was collected and stored at Ϫ20°C. The genotype of norovirus GII in the specimen was found to be norovirus GII genotype 6 (GII.6), which was revealed by RT-PCR and sequencing of the norovirus GII gene as described previously (24).A contamination test of oysters with norovirus GII and FCV f4 was conducted at Miyagi Prefecture Fisheries Research and Development Center. Three water baths were prepared, each containing 160 liters of sand-filtere...
An etiological study was conducted to clarify whether the flagellate-like cells found in histological preparations of the tunic of diseased Halocynthia roretzi (Drasche) were the causative agent of soft tunic syndrome in this ascidian. When pieces of softened diseased tunic were incubated overnight in sterile seawater, live flagellated cells, which were actively swimming in the seawater, were observed in 47 out of 61 diseased ascidians (77%), but not in moribund or abnormal individuals with normal tunics (n = 36) nor in healthy animals (n = 19). The flagellate was morphologically very similar to those observed in histological sections of the diseased tunic. By contrast, flagellates were not found in tunic pieces of healthy, moribund, and abnormal individuals that did not exhibit softening of the tunic. Light and electron microscopy revealed that the flagellate has polykinetoplastic mitochondria with discoidal cristae. The cytomorphologies of the flagellate were the same as those of the flagellate-like cells in the diseased tunic. We cultured the flagellate from the softened tunic in vitro and confirmed that the tunics of healthy ascidians, which were immersion-challenged with suspensions of the subcultured flagellates, became softened 17 d after exposure, including the final 12 d in aerated, running seawater. The occurrence of flagellates was also confirmed by incubating pieces of soft tunic from experimentally infected animals in seawater overnight. These results indicate that the flagellate is the causative agent of soft tunic syndrome. KEY WORDS: Ascidian · Halocynthia roretzi · Soft tunic syndrome · Causative agent · Kinetoplastid protist · Flagellate Resale or republication not permitted without written consent of the publisherDis Aquat Org 95: 153-161, 2011 154 that the disease is transmissible by experimental infection in which pieces of softened tunics were immersed in the rearing seawater of healthy animals. The result indicated that the causative agent was released from the affected animals, became waterborne, and infected healthy ascidians. Histology showed flagellate-like cells (10-14 × 2-3 µm) in the tunics of spontaneously or experimentally diseased ascidians, but not in the apparently healthy animals (Kumagai et al. 2010). In situ hybridization using probes for the ascidian 18S rRNA gene did not result in a signal for the gene in the flagellate-like cells, indicating the possibility that these cells are not ascidian cells but are a distinct organism (Kumagai et al. 2010). In addition, neither bacteria nor virus-like particles were observed in affected ascidians (Hirose et al. 2009, Kitamura et al. 2010, Kumagai et al. 2010. The results of the physical properties of the causative agent are consistent with the hypothesis that the flagellate-like cells are a flagellated protist, and the causative agent of soft tunic syndrome; the tunics of affected animals lost infectivity when frozen or homogenized, and the presumed infectious agent could pass through a 5.0 µm membrane filter, but not ...
The contamination of oysters with human noroviruses poses a human health risk, since oysters are often consumed raw. In this study, human norovirus genogroup II was allowed to bio-accumulate in oysters, and then the effect of high-pressure processing (HPP) on human noroviruses in oysters was determined through a polymerase chain reaction (PCR)-based method with enzymatic pretreatment to distinguish infectious noroviruses. As a result, oysters could be artificially contaminated to a detectable level of norovirus genome by the reverse transcription-PCR. Concentrations of norovirus genome in laboratory-contaminated oysters were log normally distributed, as determined by the real-time PCR, suggesting that artificial contamination by bio-accumulation was successful. In two independent HPP trials, a 1.87 log and 1.99 log reduction of norovirus GII.17 genome concentration was observed after HPP at 400 MPa for 5 min at 25°C. These data suggest that HPP is a promising process of inactivation of infectious human noroviruses in oysters. To our knowledge, this is the first report to investigate the effect of HPP on laboratory-contaminated noroviruses in oysters.
The contamination of oysters with human norovirus (HuNoV) poses a human health risk, as oysters are often consumed raw. In this study, the effect of high pressure processing (HPP) on a wide variety of HuNoVs naturally present in aqua-cultured Japanese oysters was determined through a polymerase chain reaction-based method with enzymatic pretreatment, to distinguish between infectious HuNoV. Among five batches, genogroup I. genotype 1 (GI.1), GI.2, GI.3, and GI.8 HuNoV were detected from only one oyster not treated with HPP in the fifth batch, while genogroup II. genotype 1 to 4 (GII.1 to 4), GII.6, GII.8., GII.9, GII.13, GII.16, GII.17, and GII.22 HuNoV were detected from oysters not treated with HPP in all tested batches as determined by next-generation sequencing analysis. Neither GI nor GII HuNoV was detected in the oysters of any of the batches after HPP treatment. To our knowledge, this is the first study to investigate the effect of HPP on a wide variety of HuNoVs naturally present in aqua-cultured oysters.
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