The inadequacy of E. coli
as an indicator of the viral risk associated with oyster consumption is well documented (7
) and has prompted calls for investigations of alternative viral indicators. The inadequacy of the E. coli
standards was confirmed in this study; all of our samples met the EC E. coli
end product standard (less than 230 per 100 g of shellfish) despite the fact that oysters obtained from two of the sites were implicated in outbreaks of viral gastroenteritis. It is important to note that the shellfish used in this study, like many oysters both in the United Kingdom and in other countries, were purified with commercial depuration systems prior to sale. Depuration has been shown to rapidly and effectively remove bacterial pollution indicators; however, human enteric viruses are known to be more persistent (1
; M. D. Sobsey, J. C. Murray, and G. Lovelace, Abstr. 91st Annu. Meet. Am. Soc. Microbiol. 1991, p. 300, 1991). It is not surprising, therefore, that the absence of E. coli
in depurated oysters may not guarantee a virus-free product. This is particularly evident during viral infectious disease outbreaks in which shellfish implicated on the basis of epidemiological criteria frequently comply with the bacterial end product standard, less than 230 E. coli
per 100 g (4
In this study we evaluated whether FRNA bacteriophages could be used as alternative viral indicators for shellfish. The FRNA bacteriophage contents of market-ready oysters from four producers were compared with the pollution status of the harvest areas, with reported gastroenteric illness incidents linked to products from each site during the study period, and with NLV contamination, as judged by RT-PCR. FRNA bacteriophages, like E. coli and NLV, were not present in oysters harvested at a pristine site that was free of sewage pollution and was not previously associated with gastroenteric illness. This illustrates that it is possible to obtain shellfish that do not contain FRNA bacteriophages and present a low health risk. In contrast, FRNA bacteriophages were detected, often at high levels, in market-ready oysters harvested at more polluted sites. Furthermore, the frequency and degree of FRNA bacteriophage contamination were closely associated with consumer health risk due to enteric viruses, as judged by the degree of harvest area pollution, the NLV content of shellfish, and the association with reported incidents of gastroenteric illness. These data suggest that FRNA bacteriophages, unlike E. coli, are reliable and effective indicators of the possible presence of human enteric gastroenteritis viruses in depurated market-ready oysters.
The presence of FRNA bacteriophages in depurated oysters indicated that the oysters were subject to fecal contamination in their harvest areas and that any viruses present may not have been eliminated during the depuration process. During this study this was demonstrated for oysters from site 4, the most polluted site, by detection of NLVs in market-ready oysters. NLVs were not detected in oysters from the other sites; however, the numbers of samples examined were low. Contamination of shellfish harvest areas by NLVs is probably sporadic and depends on viral circulation in the community. Like other indicator systems, the presence of FRNA bacteriophages in oysters, therefore, indicates the potential for viral contamination rather than a definitive hazard in a sample being studied. However, it is reasonable to assume that the titer of FRNA bacteriophages found in depurated oysters indicates the relative risk of viral contamination. This was confirmed in this study, in which the average levels of FRNA bacteriophages in depurated oysters were correlated with the frequency of NLV contamination determined by RT-PCR, the number of reported health incidents associated with products from each site, and the degree of harvest area pollution. Conversely, unlike E. coli, the absence of FRNA bacteriophages appears to be a reliable indicator that enteric viruses, such as NLVs, are probably absent.
Outbreaks of gastroenteritis associated with the consumption of oysters in the United Kingdom exhibit a clear seasonal trend, with outbreaks occurring predominantly during the winter months and only rarely in the summer months (Fig. ). The NLV data obtained for oysters in this study were entirely consistent with this trend. Interestingly, FRNA bacteriophage contamination in depurated oysters also exhibited a marked seasonal trend that was consistent with the high-risk period for contamination by enteric viruses. Traditionally, NLV gastroenteritis has been considered a seasonal disease; it was described in early studies as “winter vomiting disease.” However, in recent years the seasonal nature of the disease has been less consistent, and during our study period peak community infection levels occurred in late spring 1995 and early summer 1996 (12
). These peak community levels were not consistent with the period of NLV contamination of oysters observed in this study, suggesting that other factors may also be significant. Likewise, there is little evidence that FRNA bacteriophage levels in sewage effluents are different in different seasons, which makes this an unlikely explanation for the seasonal differences seen in oysters. It seems more likely that NLV and FRNA bacteriophage contents of oysters are influenced either by different winter and summer rates of virus inactivation in the environment or by seasonally dependent uptake and depuration of viral contaminants by molluscs. Since FRNA bacteriophage are resistant to UV irradiation (20
), it is unlikely that inactivation in the environment completely accounts for the dramatically decreased levels found in oysters during the summer months. Data on NLV decay in the environment is not available. A stronger possibility is that viruses are eliminated more efficiently during the mollusc depuration process in the summer. This could be associated with factors that affect mollusc metabolism, such as higher summer water temperatures or food availability. Clearly, these possibilities should be investigated further as they may have important implications for improving commercial depuration procedures used for virus removal during the winter months.
One possible criticism of FRNA bacteriophages as indicators of viral risk in oysters is that, like E. coli
, these bacteriophages are not human specific. Animal feces originating from land runoff could also cause FRNA bacteriophage contamination (21
) but may not pose a health risk due to NLVs. During this study there was little evidence for this as FRNA bacteriophage contamination could be accounted for by known sewage discharges and correlated well with health risk and the presence of NLVs. Oligonucleotide probe hybridization methods for geneotyping FRNA bacteriophages have recently been proposed for differentiating animal-associated and human-associated bacteriophage groups (5
). The application of such techniques to shellfish would facilitate differentiation of contamination from human sources and contamination from animal sources. Such techniques could be useful in investigations of sites where sewage pollution sources cannot be identified and contamination from animal feces is suspected.
In conclusion, data obtained in this study suggest that the FRNA bacteriophage content of depurated oysters sold for consumption may reflect the public health risk due to human enteric viruses more accurately than the E. coli content reflects this risk. Data obtained in this study also indicate that currently used commercial depuration practices cannot guarantee removal of human enteric viruses from oysters during the winter months.