Norovirus contamination in wild oysters and mussels in Shiogama Bay, northeastern Japan.

ABSTRACT The contamination with norovirus (NV), a causative agent of gastroenteritis, in wild Pacific oysters Crassostrea gigas and Mediterranean blue mussels Mytilus galloprovincialis was surveyed from 2005 to 2006 by RT-PCR, collecting monthly 10 ~ 20 samples for each species in Shiogama Bay (Shiogama Port), northeastern Japan. The bivalves examined were highly contaminated with NVs, especially with Genogroup 2 (G2), in winter and early spring, with the peak of 91% (G2) for oysters in April and 74% (G2) for mussels in March. The contamination rates in cultured Pacific oysters, concomitantly collected in a different bay (Onagawa Bay) in the same period of time, were much lower than those in wild oysters. When the contamination rates were compared among wild oysters collected at three sites with different distances from a sewage treatment plant, the incidence of G1 genogroup of NVs was higher in the oysters collected at sites nearer to the treatment plant, indicating that the drainage from the plant is the major virus source in this area. An improvement of virus inactivation in the treatment process is believed to be necessary to prevent viral contamination in coastal populations of bivalves.

KEY WORDS: norovirus, contamination, bioaccumulation, Crassostrea gigas, Mytilus galloprovincialis, mussels, oysters

INTRODUCTION

Gastroenteritis caused by noroviruses (NVs) has been an annoying disease problem in the world since the first recognized case in Norwalk, USA in 1968 (Kapikian et al. 1972). The illness is generally mild and characterized by nausea, vomiting, diarrhea, and abdominal cramps (Kaplan et al. 1982). The causative agent of the disease had long been called SRSV (Small Round-structured Virus), but now is classified as the member of the Genus Norovirus in the family Caliciviridae (Green et al. 2000, ICTV: http://www.ncbi.nlm.gov/ICTVdb). The investigation on the disease and its causative agent has been hampered by some difficulties (e.g., the pathogen cannot be grown in any established cell lines and there is no appropriate experimental animals Parashar & Monroe 2001), however, it has rapidly progressed, because the molecular biology of the virus (Jiang et al. 1990, Lambden et al. 1993) and molecular diagnostic methods (Jiang et al. 1992, Atmar & Estes 2001) were introduced. Norovirus gastroenteritis is a foodborne disease, although secondary person-to-person transmission is also common, and the potential transmission pathway was first recognized after a large outbreak of gastroenteritis in Australia in 1978 (Lees 2000, Parashar & Monroe 2001). Also in the USA similar large-scale outbreaks occurred in Louisiana and other states in 1993 (Kohn et al. 1995) and in Florida in 1995 (McDonnell et al. 1997). investigations of these outbreaks identified several features of shellfish-associated NV disease that had important implications for prevention: (1) the implicated shellfish were often consumed raw; (2) sewage contamination of the oyster-harvesting areas frequently preceded the outbreaks; (3) persons in widespread geographical areas could be affected because of the rapid distribution of contaminated shellfish; and (4) deputation (practice of holding oysters in tanks of disinfected water for a period of time) (Richards 1988) was not always effective in ensuring the safety of shellfish for consumption (Parashar & Monroe 2001). As an unavoidable result of the Japanese custom to eat fish and shellfish raw, NV infection constitutes an important gastroenteritis in Japan. In 2004, for example, about 1,700 gastroenteritis cases were recorded in Japan. Among these cases, 69% was caused by bacterial infections and 17% were because of viral infections. Bacterial diseases included Campylobacter, Salmonella, Vibrio, and other infections; but viral disease was composed of NV infection alone (MHLW: http:// www.mhlw.go.jp/topics/syokuchu). Thus the disease cannot be a negligible problem and a good number of studies have been made on this disease in various fields of sciences related to medicine and food/environmental hygiene, however, few investigations have been performed from the standpoints of shellfish biology and aquaculture in Japan (Muroga & Takahashi 2005). Now we are making a survey of NV contamination in cultured oysters, but here in this study, we report our results of the first survey on wild oysters and mussels that was made preliminarily to confirm the presence of NVs in coastal waters impacted by drainage from a city sewage treatment plant.

MATERIALS AND METHODS

Animals

Wild Pacific oysters (Crassostrea gigas Thunberg, 1793) and Mediterranean blue mussels (Mytilus galloprovincialis Lamarck, 1819) were sampled every month at a landing pier in Shiogama Port (See Fig. 1) from March 2005 to January (mussels) or March (oysters) 2006. At each sampling, 10-20 shellfish for each species were collected and transferred to our laboratory in a cool box, and target tissues (digestive diverticula including stomach) were immediately removed from shucked oysters and mussels. The average shell height, length, and width of sampled oysters and mussels were 66 mm x 42 mm x 25 mm and 60 mm x 33 mmx 23 mm, respectively. For comparison, similar number of cultured Pacific oysters (average shell height, length, and width were 124 mm x 61 mm x 34 mm) were also collected monthly in Onagawa Bay (see Fig. 1) in almost the same period of time, and tissue samples were removed from them in the same way as in the wild oysters. Records of water temperature in Shiogama Bay were obtained from the Office of Eastern Urado Fishermen's Union of Shiogama City, and those in Onagawa Bay were from the Marine Field Station of Field Science Center, Tohoku University. To estimate the contamination source of NVs for the sampling sites in Shiogama Bay, wild Pacific oysters were collected at two additional sampling stations (St. 1 and St. 2) on March 16, 2005 in a ditch (width is approximately 30 m) connecting a city sewage treatment plant (Miyagi Central-South Sewage Treatment Plant) and Shiogama Port. The distances from the discharge point of the plant to the three sites, St. 1, St. 2, and the monthly sampling site in Shiogama Port (St. 3) were 0.6 km, 2.5 km, and 3.8 km, respectively.

[FIGURE 1 OMITTED]

Detection of NVs From Tissue Samples by RT-PCR

Noroviruses were detected in bivalves using the RT-PCR method recommended by the Ministry of Health, Labor and Welfare, Japan (HYPERLINK "http://www/mhlw.go.jp/ topics/syokuchu/kanren/kanshi/dl/031105-1a.pdf" http:// www/mhlw.go.jp/topics/syokuchu/kanren/kanshi/dl/031105la.pdf) with some modifications.

Pretreatment of Samples

The removed digestive diverticulum sample was put into a disinfected polyethylene tube with 2 stainless-steel beads, and distilled deionized water was added to the tube in the proportion of 1 mL per 1 g of the tissue sample. Then, the sample was homogenized by Micro Smash TM SM-100 (Tomy, Tokyo) for 1 min at 4,500 rpm (Ueki et al. 2005). The homogenized sample was centrifuged at x 9,000 g for 10 min and the supernatant was submitted to RNA extraction.

RNA Preparation

RNA was extracted from the treated samples by using QIAamp Viral RNA Mini Kit (QIAGEN, Tokyo) by following the manufacturer's instruction. The extracted RNA samples were stocked at -30[degrees]C until the next procedure.

DNase Treatment and Reverse Transcription 

The thawed RNA templates were treated with DNase (RQ1 DNase, Promega, Tokyo) and reverse transcribed in the RT reaction mixture shown in Table 1 at 42[degrees]C for 1 h. The RT enzyme was inactivated at 99[degrees]C for 5 min and the RT mixture was stored at 4[degrees]C until the next PCR step.

PCR

Amplification of the cDNA sample (RT mixture) was made in PCR reaction mixture shown in Table 2. The PCR profile was run with 1 cycle of 94[degrees]C for 3 min, 40 cycles of 94[degrees]C x 1 min--50[degrees]C x 1 min-72[degrees]C. 1 min, and 1 cycle of 72[degrees]C for 15 min. For the RT reaction, primers G1-SKR and G2-SKR were used for Genogroups 1(G1) and 2(G2), respectively (Kojima et al. 2002). For the first PCR reaction, primer sets COG1F (forward)/ G1-SKR (reverse) and COG2F/G2-SKR were used for G1 and G2, respectively (Kageyama et al. 2003). For the nested PCR, primer sets, G1-SKF/NewG1R and G2-SKF/NewG2R were used for G1 and G2, respectively. NewG1R and NewG2R were designed in the present study for easier determination of the products in nested PCR procedure. Sequences of these newly designed primers are as follows: NewG1R: ACATCAC CGGGGGTATTATTTGG; NewG2R: GGCTTGTACAA AATTATTTCTAA. The lengths of the expected products from the nested-PCR were 252 bp for G1 and 216 bp for G2. Ten microliters of the products from the nested-PCR were analyzed by 1.5% agarose gel electrophoresis and visualized by SYBRSafe dye (SYBRSafe DNA Gel Stain, Invitrogen, Tokyo). Each run included negative and positive controls.

Sequencing of RT-PCR Products:

The representative samples of DNA products from nested PCR were sequenced by an analytical agent (Bio Matrix Research, Inc., Kashiwa, Japan).

RESULTS

With the exception of a few samples (less than 10% of the tested samples), the sequenced samples of DNA products in nested PCR were confirmed to be part of the NV genes. We are now comparing the sequence variations in G1 and G2 of NVs detected from wild oysters with those found in cultured oysters, including some from oyster farms still under investigation. Seasonal changes in detection rates of NVs from wild Pacific oysters collected in Shiogama Port are shown in Table 3 and Figure 2 with changes in average water temperature in Shiogama Bay. High detection rates, especially for G 2, were recorded in March (80%, G2) and April (91%, G2) 2005 when the survey was started. After May, NVs were not detected in any samples until December. All the samples examined in February and March 2006 were contaminated with G2 of NVs. Figure 3 (Table 3) shows the seasonal changes in detection rates of NVs from wild mussels in Shiogama Port. As in the case of wild oysters, NVs, especially G2, were detected at high rates in the early spring (March and April) and in the winter (December and January), however, differing from the results of oysters, NVs (G2) were constantly detected in one sample (detection rate was 10%) throughout the summer (June to August). NVs were also detected from cultured oysters in Onagawa Bay in the winter (December to February) (Table 3 and Fig. 4). However, the contamination rates were much lower than those of wild oysters. The monthly average water temperature fluctuated between 3.1[degrees]C (February 2006) and 26.2[degrees]C (August 2005) in Shiogama Bay (Figs. 2, 3), and between 5.7[degrees]C (March 2006) and 22.8[degrees]C (August 2005) in Onagawa Bay (Fig. 4). The range between the lowest and the highest temperatures in Shiogama Bay was greater than that in Onagawa Bay, however, the seasonal changes were quite similar to each other. Figure 5 shows the result of NV detection made in March 2005 to compare the contamination rate of oysters in the three sampling sites with different distances from the sewage treatment plant. The G1 genogroup was detected in the higher rates in the sampling sites nearer to the plant, but the contamination rate of G 2 was higher at St. 3 (Shiogama Port) than at St. 2.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

DISCUSSION

The two sampling sites, Shiogama Bay and Onagawa Bay, are both in Miyagi Prefecture, where oyster culture constitutes important fisheries industry producing approximately 50,000 metric tons of oysters with shell every year (second to Hiroshima Prefecture in Japan). Thus NV contamination in cultured oysters is a serious problem in this prefecture. According to the results of our investigation, wild Pacific oysters and Mediterranean blue mussels were highly contaminated with NVs, especially in winter and early spring. It is natural that the NV contamination rate in cultured oysters in Onagawa Bay were much lower than those in wild oysters in Shiogama Port, because Onagawa Bay itself is less polluted than Shiogama Bay, and the culture site was remote from the inner part of the bay. The heavy contamination with NVs in wild oysters and mussels indicate that wild coastal populations of bivalve molluscs contribute to removal of human-pathogenic viruses from environmental waters. Cultured Pacific oysters collected in winter in western Japan were confirmed to be contaminated with NVs in a previous study (Nishida et al. 2003), however, no data on seasonal changes in NV contamination in bivalves have been published in Japan. Le Guyader et al. (2000) made a three-year investigation on NVs and other human enteric viruses in Pacific oysters and Mediterranean blue mussels cultured in France. They reported higher detection rates of NVs in winter (November to January). Burkhardt III & Calci (2000) also demonstrated that oysters preferentially accumulated [F.sup.+] coliphage, an enteric viral surrogate, to their greatest levels from late November through January, and noted three factors for the phenomenon that shellfish-associated viral illness frequently occurs in winter: (1) the level of prevalence of enteric viral pathogens in wastewater is higher in winter; (2) viral pathogens can survive in the estuarine environment for a longer period in winter; and (3) the ability of shellfish to selectively accumulate viruses becomes greater in winter. The major reason for the high contamination rates of NVs in winter in wild oysters and mussels shown in the present study must be the prevalence of NV infection in human populations, because NV infection most frequently occurs in winter also in Japan. The last factor raised by Burkhardt III & Calci (2000) is interesting from the standpoint of physiology of bivalve molluscs. They noted that high glycogen content of shellfish in winter might influence the ability of bioaccumulation of shellfish. In addition to this, the following mechanism seems to be involved: the lowered defense factors including various enzymatic activities at lower water temperatures will hamper the activities of shellfish to inactivate and eliminate contaminating viruses. Throughout the three surveys (Figs. 2-4), the genogroup G 2 of NV always dominated over G 1. This tendency is in accordance with the results of previous studies made against cultured oysters in Japan (Nishida et al. 2003, Ueki et al. 2005). It should be stressed that G 2 was detected constantly from wild mussels even in July and August 2005, which perfectly coincided with the confirmed occurrences of NV gastroenteritis in Miyagi Prefecture in July (one case) and August (two cases) 2005 (Kikuchi et al. 2006). This demon strates that the contamination of NVs in coastal bivalves is a good indicator of occurrence of NV gastroenteritis not only in winter but also in other seasons. The declining of contamination rate with NVs, especially G1, in wild oysters collected in the sampling sites farther from the sewage treatment plant (Fig. 5) suggests that the drainage water from the plant is the source of NVs that contaminated coastal wild oysters in Shiogama Port. However, contrary to our expectation, the contamination rate with G2 was higher at St. 3 (Shiogama Port: farthest site from the plant) than St. 2 (midway between St. 1 and St. 3), indicating the presence of other virus source(s) in the bay. The sewage treatment plant involved in this study implemented a disinfection process with hypochlorite in the final stage of the treatment, confirming the coliforms being less than 30 cells/mL in drainage water. The results of our study indicate that a considerable amount of NVs were released from the plant without inactivation. This is not an unusual case, because Ueki et al. (2005) detected NVs from treated wastewater from a different treatment plant in Miyagi Prefecture. A similar situation of NV discharge from a sewage treatment plant was demonstrated by relocation experiments with oysters (possibly Crassostrea virginica, but the species name was not given) in a polluted area in the Gulf of Mexico (Shieh et al. 2003). Conventional wastewater treatment systems might be insufficient to inactivate human enteric viruses like NVs. Thus, as suggested by Ueki et al. (2005), an improvement for virus inactivation in the treatment process must be made to prevent contamination with these viruses in coastal and cultured oysters.

ACKNOWLEDGMENTS

The authors thank Mrs. Y. Ueki and N. Yamaki of the Miyagi Prefectural Institute of Public Health and Environment for their helpful advices on RT-PCR detection of NVs. This study was partly supported by a grant from Japan Society for the Promotion of Science (Grant number: 17580152).

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FUMIHITO MAEKAWA, YUKIE MIURA, AKIHIRO KATO, KEISUKE G. TAKAHASHI * AND KIYOKUNI MUROGA

Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan

* Corresponding author: E-mail: [e-mail]

TABLE 1. Composition of the reaction mixture
for RT reaction.

Components                       Volume

5 x RT PCR Buffer (Invitrogen)   3 [micro]L
RNase DNase free
  distilled water                2.83 [micro]L
0.1 M DTT (Invitrogen)           1 [micro]L
DNTP (Invitrogen) (10 mM)        1 [micro]L
RNase OUT (Invitrogen)           0.67 [micro]L
Superscript II RT (Invitrogen)   1 [micro]L
Reverse Primer (25 pM)           0.5 [micro]L
DNase-treated RNA sample         10 [micro]L
Total                            20 [micro]L

TABLE 2. Composition of the reaction mixture
for first and nested PCR.

Components                           Volume

RNase DNase free distilled water     33.75 [[micro]L.sup.1]
10 x Ex Taq buffer (Takara)           5 [micro]L
dNTP (Takara) (2.5 mM)                4 [micro]L
Ex Taq Hot Start version (Takara)     0.25 [micro]L
Forward primer (25 [micro]M)          1 [micro]L
Reverse primer (25 [micro]M)          1 [micro]L
Template for 1st PCR or nested PCR    5 [[micro]L.sup.2]
Total                                50 [micro]L

(1) 36.75 pL for nested PCR.

(2) 2 [micro]L for nested PCR.

Others were common for the first and the nested PCRs.

TABLE 3. Detection of noroviruses (GI, G2) from wild Pacific
oysters and Mediterranean blue mussels collected in Shiogama
Port and from cultured Pacific oysters in Onagawa Bay.

                            2005

               Jan.         Feb.           Mar.

Wild Oysters
  G1          NE (1)         NE        27 (4/15) (2)
  G2            NE           NE         80 (12/15)

Wild Mussels
  G1            NE           NE          26 (5/19)
  G2            NE           NE          74(14/19)

Cultured Oysters
  G1        30 (3/10)     0 (0/15)       0 (0/10)
  G2         0 (0/10)     0 (0/15)        0(0/10)

                            2005

               Apr.          May           Jun.

Wild Oysters
  G1        17 (4/23)      6(1/16)       0 (0/12)
  G2        91 (21/23)     0(0/8)        0 (0/12)

Wild Mussels
  G1        10 (0/10)     20 (2/10)       0(0/10)
  G2        60 (6/10)     0 (0/10)       10(0/10)

Cultured Oysters
  G1         0(0/10)      0 (0/10)        0 (0/9)
  G2         0 (0/10)     0 (0/10)        0(0/9)

                            2005

               Jul.         Aug.           Sep.

Wild Oysters
  G1         0 (0/13)     0 (0/15)        0 (0/9)
  G2         0(0/13)      0 (0/15)        0 (0/9)

Wild Mussels
  G1         0(0/10)       0(0/10)       0 (0/10)
  G2        10 (0/10)     10 (0/10)      0 (0/10)

Cultured Oysters
  G1         0(0/10)      0 (0/10)        0(0/10)
  G2         0(0/10)       0(0/10)       0 (0/10)

                            2005

               Oct.         Nov.           Dec.

Wild Oysters
  G1         0(0/14)      0 (0/10)       20 (3/15)
  G2         0 (0/14)     0 (0/10)       40 (6/15)

Wild Mussels
  G1         0(0/10)      0 (0/10)       50 (5/10)
  G2         0 (0/10)     0 (0/10)       60(6/10)

Cultured Oysters
  G1         0 (0/10)     0 (0/10)       0 (0/10)
  G2         0(0/10)      0 (0/10)       20(2/10)

                            2006

               Jan.         Feb.           Mar.

Wild Oysters
  G1        40 (6/15)     13 (2/25)      30 (3/10)
  G2        87 (13/15)   100 (15/15)    100(10/10)

Wild Mussels
  G1         0(0/10)         NE             NE
  G2        50 (5/10)        NE             NE

Cultured Oysters
  G1         0(0/13)      0 (0/15)       0 (0/15)
  G2         8 (1/13)     7 (1/15)       0 (0/15)

(1) NE: not examined.

(2)

Percentage (number of shellfish positive
for NV/number of shellfish examined).

Figure 5. The detection rate of NVs (G1, G2) in wild oyster (C. gigas)
by RT-PCR at three stations in a ditch connecting the sewage treatment
plant to Shiogama Port (St. 3) in March 16, 2005.

                              G1         G2
St. 1 (0.6 Km (*2)        14/15 (*1)    13/15
St. 2 (2.5) Km)            7/14          9/14
St. 3 (3.8 Km)             4/15         12/15

(*1) Number of oysters positive for NV/number of oysters examined

(*2) Distance fromthe sewage treatment plant

Note: Table made from bar graph.