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(43) Selikoff, I. J.; Hammond, E. C.; Seidman, H. Ann. N.Y. Acad. Sci. 1979,330,91-116. (44) Newhouse, M. L.; Thompson, H. Br. J . Znd. Med. 1965,22, 261-269. (45) Churg, A.; Warnock, M. L. Am. Rev. Respir. Dis. 1980,122, 669-678. (46) Hughes, J. M.; Weill, H. Am. Rev. Respir. Dis. 1986,133, 5-13. (47) Mossman, B. T.; Bignon, J.; Corn, M.; Seaton, A.; Gee, J. B. L. Science 1990,247, 294-301. (48) Lippmann, M. Environ. Res. 1988, 46, 86-106. (49) Fed. Regist. OSHA SI FR 22612; June 20, 1986. (50) Stanton, M. F.; Layard, M.; Tegeris, A. J. Natl. Cancer Znst. 1981, 67, 965-975. (51) Davis, J. M. G.; Addison, J.; Bolton, R. E.; Donaldson, D.; Jones, A. D.; Miller, B. G. Carcinogenesis 1985,6,667-674.
(52) Davis, J. M. G.; Addison, J.; Bolton, R. E.; Donaldson, K.; Jones, A. D.; Smith, T. Br. J . Explor. Pathol. 1986, 67, 415-430.
Received for review January 30,1991. Revised manuscript received September 3,1991. Accepted September 17,1991. This research was supported through the US.EPA Office of Research and Development Indoor Air Program, Research Triangle Park, NC 27711. Although the research described in this article has been funded wholly or i n part by the United States Enuironmental Protection Agency, it has not been subjected to Agency review and therefore does not necessarily reflect the views of the Agency, and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
Contamination of Shellfish by Stool-Shed Viruses: Methods of Detection Fred P. Williams, Jr.” and G. Shay Fout Virology Branch, Microbiology Research Division, Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268 ~~
Molluscan shellfish, being filter feeders, are readily contaminated with the stool-shed viruses present in human sewage. Consumption of such virus-contaminated shellfish has caused outbreaks of hepatitis A and viral gastroenteritis in the United States and in other countries. As coastal communities continue to develop and expand, shellfish habitats are increasingly at risk for such exposure. Effective methods are needed to examine shellfish for virus contamination. This review describes methods that are currently available and reports on recent method developments. In the past, methods depended upon slow cell culture procedures for the assay of virus. Now, more rapid assay procedures are becoming available as advanced techniques of biotechnology are beginning to be applied. However, despite 30 years of method development, there remains little agreement on a “standard method” to be used for routine analysis of shellfish for virus.
For the environmentalchemist, many of today’s analyses involve rapid, highly sensitive procedures that incorporate the latest in high technology hardware and software. For the environmental virologist, whose procedures depend heavily on the use of cell cultures, analytical methods have been comparativelyless rapid, less standardized, and more labor intensive. However, this situation is changing. Virologists are increasingly applying new techniques of biotechnology to environmental analyses. This review of methods for detecting viruses in shellfish reports on an area that is now being impacted by such developments. Molluscan shellfish, being filter feeders, are readily contaminated through exposure to domestic sewage. Shellfish habitats in shallow coastal waters are increasingly at risk for such exposure because human coastal communities continue to develop and expand. Although human stool-shed viruses found in sewage are not known to replicate in or to cause disease in shewish, they present a clear health hazard to humans consuming shellfish contaminated by these viruses. Two human illnesses caused by viruses, hepatitis A and viral gastroenteritis, have been associated with the consumption of contaminated shellfish. Hepatitis A, which has an incubation period of 15-50 days, is characterized by fever, malaise, nausea, anorexia, abdominal discomfort,
dark urine, and jaundice (1). In many infected children and some adults, the disease may be asymptomatic. Occasionally, prolonged disability or death occurs, especially in the elderly. Viral gastroenteritis (noninfantile) caused by agents such as Norwalk virus, Snow Mountain agent, and other small round structured viruses (SRSVs) includes as symptoms nausea, vomiting, diarrhea, and abdominal cramps (2). Symptoms experienced less often include headache, fever, chills, and myalgias. The symptoms last 12-60 h and follow an incubation period of 24-48 h. In the elderly, symptoms may last longer.
Viruses and Disease Outbreaks Potentially, over 100 different viruses shed in human stools could contaminate shellfish exposed to polluted waters. To date, relatively few have been implicated in outbreaks of shewish-borne illness. Viruses that have been implicated (and these to varying degrees) include the following: hepatitis A virus (HAV) (3,4), Norwalk virus (5-7), Snow Mountain agent (8), nonspecified SRSV (9, IO), cockle agent (9, I I ) , nonspecified parvovirus (9),astrovirus (9,12), and calicivirus (9,13). Except for HAV, these viruses were all associated with gastroenteritis outbreaks. There is some evidence that an additional hepatitis-producing virus (enterically transmitted non-A, non-B hepatitis virus) may be responsible for sporadic cases of shellfish-borne hepatitis (14, 15). To protect the shellfish consumer from disease, standards based on the coliform group of bacteria have been established. Shellfish harvesting is restricted in areas where the harvesting waters exceed 70 total coliforms or 14 fecal coliforms per 100 mL (16). In addition, the indicator levels in shellfish at the wholesale market level must not exceed 230 fecal coliforms/100 g and a standard plate count of 500000/g of shellfish meat (17).In spite of these standards, during 1961-1984, there were approximately 1400 reported cases of hepatitis A in the United States due to shellfish consumption (1419). For the period 1934-1984, there were 6000 reported cases of shellfish-associated gastroenteritis. The annual number of cases of these shellfish-borne illnesses varies considerably and may follow a cyclic pattern. The number of cases of hepatitis A has declined significantly in the United States
Not sublect to U S . Copyright. Published 1992 by the American Chemical Society
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Fyure 1. Electron micrographs of
Norwalk and hepatitis A
vlruses.
since the 196Os, while the number of cases of gastroenteritis has increased. Although it is not known how many of the reported cases of shellfish-borne gastroenteritis were caused by viruses, it is likely a significant percentage. In 1982, over 100 shellfish-borne outbreaks with a total of 1017 cases of gastroenteritis were reported in New York State. Of the 95 outbreaks where sufficient information was available, 76 proved clinically and epidemiologicallyconsistent with a Norwalk virus-like pattern of illness. In addition, lahoratory examination of samples from seven of the outbreaks confirmed Norwalk virus involvement in five of the seven outbreaks (7). Norwalk virus also caused a 1978 Australia-wide shellfish-borne outbreak that resulted in 2 ~ 7 0 0 individual 0 cases of illness (20-22). In England and Wales, 1981-1983, viral illness (both hepatitis A and viral gastroenteritis) accounted for over 60% of the shellfish-borne outbreaks (23). In many outbreaks the causative agent is never identified, and it is probable that many of these are due to viruses that are not detected. Various factors have contributed to the failure to confirm a viral etiology for shellfish outbreaks of illness. One factor has been that tests to detect such agents as Norwalk virus and Snow Mountain agent have not been widely available. Tests for these agents have been developed at several research centers and have been effectively used to show serological evidence of viral infection in afflicted individuals. These tests have involved the use of immunoassays or electron microscopy (EM) since sensitive cell culture techniques for these viruses remain unavailable. Unfortunately, serum specimens for such testing are not always obtained during outbreak investigations. While stool specimens from afflicted individuals can be successfully tested for these agents, specimens must be promptly collected during the acute phase of illness when the viruses are being shed in detectable numbers. Due to the limited duration of viral gastroenteritis, the collection of suitable specimens can be difficult. Direct tegting of suspect shellfish for these agents is less successful than for stool samples. This is most likely due to the inadequacy of the EM and immunoassay methods to detect low numbers of these viruses. Direct EM requires a virus titer of -106 particles/mL. Although the sensitivity can be slightly enhanced through use of immune serum or ultracentrifugation, EM is still not comparable to sensitive cell culture methods. Detection limits for the immunoassays developed for these viruses are not certain since these viruses are not readily quantifiable in cell culture. However, even amplified immunoassays can require lo5virus particles/mL for detection. 690
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From nonoutbreak studies of other stool-shed viruses more readily detected in low numbers, it is evident that virus titers in contaminated shellfish are indeed low. In selected studies (24-27), virus titers in contaminated shellfish averaged 22 infectious units/100 g of shellfish tissue with a range of 0.2-224. Among recent occurrences, the largest outbreak of shellfish-borne hepatitis A in the United States since 1973, and the largest ever in Florida, occurred in 1988 (28). The outbreak afflicted 61 individuals residing in five U.S. states. The outbreak was traced to the consumption of oysters and scallops harvested from northwestern Florida coastal waters. The sheufsh were apparently harvested from beds outside approved waters, and 60 of the 61 afflicted individuals had consumed the shellfish raw. Also in 1988, an epidemic of almost 300000 cases of hepatitis A occurred in Shanghai,China. The initial cases were reported to have involved young adults who had eaten undercooked freshwater clams (29). In Italy, from September of 1988through January of 1989, an outbreak of 47 cases of hepatitis A occurred in a city located in the Umbria Region. Both the consumption of raw mussels and the consumption of mineral water were associated with the outbreak (30). These occurrences demonstrate the risk, in the United States and elsewhere, in consuming raw or undercooked shellfish. Early Shellfish Studies Vial studies on shellfish have been performed for many years. In Europe, as early as 1958, Crovari reported on the uptake and elimination of poliovirus by the mussel, Mytilus edulis (31). In 1964, Hedstrom and Lycke, responding t o shellfish-borne outbreaks of hepatitis in Sweden and the United States, reported on poliovirus uptake and elimination in oysters (32). They also examined the effectiveness of chlorination for oyster decontamination. In the United S t a h in 1965, Metcalf and Stiles (33)modified a method for the isolation of enteroviruses from sewage and used it to detect viruses in oysters (Crassostrea uirginica) harvested from polluted waters. The authors additionally observed significant retention of virus by oyster specimens experimentally seeded with virus in the laboratory. In the years that followed, numerous other shellfish studies were undertaken. In many, shellfish were experimentally seeded with laboratory-strain enteric viruses and aspects of virus uptake, tissue distribution, and virus elimination examined. In some studies, shellfish taken directly from environmental waters were examined for the presence of naturally accumulated enteric viruses. Progress in the detection and occurrence of viruses in shellfish was such that by 1978, Gerba and Goyal were able to review a substantial body of scientific literature in this field (34). Methods for Processing Shellfish Since the earliest studies, a variety of methods have been used to recover virus from shellfish. Numerous method modificationsand new techniques have been (and continue to be) reported. In 1982, Sobsey (35)categorized shellfish methods into three main types according to the procedures used. These types included methods incorporating (a) an adsorption-elution-precipitation procedure, (b) an elution-precipitation procedure, or (c) a filtration-hydroextraction procedure. Such procedures were reported to be capable of recovering a t least 50% of the viruses present in shellfish. Specific methods or schemes, representing each of the three types, were presented in ref 36. A specific method,
Table I. Processing Shellfish for Viruses: Basic Steps”
method type extraction-concentration 1. homogenization of shellfish meat 2. extraction of virus from shellfish solids
elution medium used polyelectrolyte added conductivity adjusted centrifugation to sediment the virus-free solids 3. concentration of virus from supernatant beef extract added pH lowered to promote precipitation centrifugation to sediment virus-precipitate complexes 4.resuspension of sedimented virus in small volume of sodium phosphate solution pH adjusted 5. assay OS virus in final volume
adsorption-elution-concentration 1. homogenization of shellfish meat 2. adsorption of virus to shellfish solids
pH lowered conductivity adjusted centrifugation to sediment virus-adsorbed solids 3. extraction of virus from the shellfish solids elution medium used centrifugation to sediment virus-free solids 4. concentration of virus from supernatant pH lowered to promote precipitation centrifugation to sediment virus-precipitate complexes 5. resuspension of sedimented virus in small volume of sodium phosphate solution pH adjusted polyelectrolyte added final centrifugation to sediment remaining material that may interfere with assay 6. assay of virus in final supernatant
Reference 37.
however, was not recommended as these methods had not been systematically evaluated in collaborative study. In 1987, Sobsey (37) again reviewed methods for virus recovery from shellfish. This time, methods were reclassified into only two basic categories: (a) extraction and extraction-concentration methods and (b) adsorptionelution-concentration methods. Two specific methods or schemes were presented for each category. These methods were recommended on the basis of their simplicity, economy, and (for at least one of the methods) collaboratively evaluated effectiveness. Basic steps in the recommended methods are summarized in Table I. Extraction-Concentration. Briefly, the two recommended methods of extraction-concentration incorporate the following procedures (for full details, see ref 37). In one method, clams or oysters are shucked to obtain 100 g of shellfish meat. The meat is then homogenized for 1 min in a blender. An eluting medium of 0.09 M glycineNaOH, pH 9.5, is mixed with the homogenate and a polyelectrolyte solution (e.g., Cat-floc) added to a final concentration of 0.02% for clams and 0.1% for oysters. Conductivity is adjusted for an equivalent of
