gastroenteritis in 1978, and showed that only Georges River oysters caused. Norwalk virus infections and that depuration as carried out in 1979 was not.
AJEBAK 59 (Pt.2) 219-228 (1981)
© NORWALK VIRUS GASTROENTERITIS IN VOLUNTEERS CONSUMING DEPURATED OYSTERS by G. S. GROHMANN*, A. M. MURPHY*, P. J. CHRISTOPHERf, E. AUTYJ AND H. B. GREENBERG§ (From the *Virology Department, Institute of Clinical Pathology and Medical Research, fHealth Commission of New South Wales, the $New South Wales State Fisheries and the §Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.) (Accepted for publication January 9, 1981) Summary. Following the widespread outbreaks of oyster-associated gastroenteritis which occurred throughout Australia in 1978, several programmes were introduced to minimise the occurrence of further outbreaks. One programme included the depuration (purification) of oysters and the use of human volunteers to testconsume samples from batches of depurated oysters before their sale to the public. Oysters from the Georges River and Brisbane Waters were test-consumed from December, 1978, to August, 1979. None of the volunteers was ill after consuming Brisbane Waters oysters but 52 reported ill after eating Georges River oysters. The predominant symptoms were nausea, vomiting and diarrhoea with an average incubation period of 42 hours. Recovery was usually complete in 36-48 hours. Of the 52 illnesses reported 31 (60%) occurred in two particular weeks ending July 1st and 22nd when rates of 18 3% and 7 8% were reported. The average illness rate for the remainder of the period under study was only 1 %. Norwalk virus was found in 8 of 25 (32%) stools, and antibody increases demonstrated in seven of ten paired sera, giving an overall diagnostic rate for Norwalk infection of 37 0% for these two peak periods. Heavy rain preceded these two weeks in which the illnesses occurred. No evidence of Norwalk infection was found at any other time. These studies confirmed the epidemiological findings of the major outbreak of gastroenteritis in 1978, and showed that only Georges River oysters caused Norwalk virus infections and that depuration as carried out in 1979 was not entirely satisfactory.
INTRODUCTION Several widespread outbreaks of gastroenteritis occurred in Australia during 1978 which epidemiological investigations linked to the consumption of Sydney rock oysters {Crassostrea commercialis). The largest outbreak, involving well over 2,000 persons throughout Australia, occurred during June/July, 1978 (Murphy et al, 1979) and a second localised outbreak, * Address for reprints: A. M. Murphy, P.O. Box, Wentworthville, N.S.W. 2145.
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involving at least 150 persons, occurred during December, 1978 (Linco and Grohmann, 1980). Circumstantial evidence indicated that most of the suspect oysters were harvested from the Georges River in Sydney and microbiological investigations showed that the oysters were contaminated with sewage. It was further shown that Norwalk virus, a known cause of gastroenteritis, was the predominant pathogen in both outbreaks. Seroiogical studies using immune electron microscopy and radioimmunoassay techniques confirmed the Norwalk virus aetiology, as over 70% of patients showed a significant seroresponse to Norwalk virus (Grohmann et al, 1981). Following these episodes of oyster-associated food poisoning the Health Commission of N.S.W. and the N.S.W. State Fisheries introduced several programmes to minimise the possibility of further outbreaks. These included: (a) monitoring bacterial levels in oysters and waterways to indicate the level of pollution, (b) depuration (purification) of oysters by either natural or artificial means; natural depuration being the immersion of oysters into pollution-free waters for 7 days, and artificial depuration being the immersion of oysters into special tanks in which the water is recycled over ultraviolet light or ozone added, (c) laboratory research into the optimum conditions for removal of virus particles from the Sydney rock oyster by depuration, and (d) the use of human volunteers, as an interim measure, to test-consume samples from batches of depurated oysters before their sale to the general public. The present report relates to the fourth of these programmes and records that a number of volunteers suffered symptoms of gastroenteritis after consuming oysters which had been treated by depuration.
MATERIALS AND METHODS Testing of oysters All oysters used in this study were from the Georges River and Brisbane Waters farming areas, both of which were implicated in the Australia-wide 1978 outbreak. The area surrounding the former is heavily urbanised whereas the latter is situated just beyond greater metropolitan Sydney with much of the adjacent land being used for week-end cottages. All oysters tested by the volunteers underwent either natural or artificial depuration. Usually 5 volunteers tested each batch with each volunteer consuming a minimum of 6 oysters. The majority of the volunteers were public servants from several N.S.W. Government departments. AH volunteers were requested not to eat oysters for 72 h prior to the consumption of the test oysters. Serum specimens were collected from all volunteers prior to commencement of the programme and 'convalescent' sera and 'acute' faecal specimens were obtained in the event of illness. The number of volunteers taking part each week varied from 40 to 129, and generally the same individuals took part each week but those suffering gastroenteritis were withdrawn from the programme permanently. Any volunteer falling ill was asked to fill in a questionnaire indicating the incubation period, symptoms of illness and foods eaten just prior to testing the oysters.
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Specimens Forty-four 'acute' stool specimens were collected from ill volunteers; 32 were collected within 48 h of the onset of illness and 12 were collected after 48 h of the onset of illness. In addition, twenty sets of paired sera were obtained from individuals who were ill. The preinfection sera were collected prior to the consumption of oysters and convalescent sera between 2-8 weeks after the onset of illness. Stool and serum specimens were also collected from a further 9 volunteers who were not ill after testing oysters; from each of these volunteers a stool and serum specimen was collected prior to, within 48 h of, and 6 weeks after the consumption of oysters. Five stool specimens were also obtained from individuals not directly involved in this study but who became ill after eating oysters in restaurants. Bacteriology Samples of all the faecal specimens (76) were forwarded to the Bacteriology Department, Institute of Clinical Pathology and Medical Research, Westmead Centre, Sydney, New South Wales, where they were examined for bacteria commonly responsible for food poisoning. Cultures from 19 specimens yielding predominantly Escherichia coli colonies were sent to the School of Agriculture, La Trobe University, Victoria, for the detection of enterotoxigenic strains. Virus isolation in cell cultures Clarified 20% suspension of all faecal specimens were inoculated into cell cultures and observed for cytopathic effect as previously described (Murphy et al., 1979). Specimens positive for viruses by electron microscopy were also inoculated into suckling mice which were observed for 10 days for signs of paralysis. Electron microscopy techniques Faecal specimens. Faecal specimens were examined for the presence of viruses by electron microscopy (EM) as previously described (Murphy et al., 1979). Norwalk virus was identified by immune electron microscopy (IEM) and a single set of 'pre-infection' and 'convalescent' human sera, previously shown to seroconvert to Norwalk virus, was used. A 0 1 ml volume of 'convalescent' serum (diluted 1:10 in phosphate-buffered saline) was mixed with a 0-5 ml aliquot of concentrated semi-purified test faecal extract. The mixture was allowed to react, then ultracentrifuged and examined by EM. Faecal specimens found to contain virus-antibody complexes were re-examined using the pre-infection serum known to be free of Norwalk antibody. Virus particles were identified as Norwalk virus if they reacted with antibody from the convalescent serum and not with the pre-infection serum. Sera. Paired sera from 20 patients were tested by IEM to the 27-30 nm Norwalk virus particles and the 22-25 nm virus-like particles detected in patients from the 1978 outbreak of oysterassociated gastroenteritis. Faecal specimens containing these viruses were semi-purified and used as a source of antigen. All test sera were diluted 1:10 in phosphate-buffered saline (PBS pH 7 2) and 0 1 ml of serum was reacted with 0-5 ml of antigen. Immune aggregates were pelleted and examined by EM. All specimens were examined under code and were scored on a 0 to 4 scale according to the amount of antibody present, 0 denoting no antibody present on the virus particles and ratings of 1,2,3 and 4 denoting increasing amounts of antibody present. A difference of 1 between paired sera indicates a significant seroresponse (Kapikian et al., 1972). All specimens for EM were placed on 400 mesh Parlodion carbon-coated grids, stained with phosphotungstic acid (pH 7 0) and examined at 80 kv with a Philipps EM400G electron microscope at a plate magnification of 48000. At least 5 intact grid squares were examined per grid.
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Radioimmunoassay Aliquots from the 20 sets of paired sera were shipped in dry ice to the Laboratory of Infectious Diseases at the National Institutes of Health, Maryland, U.S.A. Serum specimens were tested, under code, for the presence of Norwalk virus antibody, by an established and sensitive micro-radioimmunoassay (RIA) blocking test as described by Greenberg et al (1978).
RESULTS Illnesses in volunteers Depuration of oysters was commenced in October, 1978, and monitoring of the system was carried out using volunteers from December, 1978, to August, 1979. Batches of oysters were tested weekly by the volunteers for 32 weeks. All oysters consumed by the volunteers were below the legal limit of bacterial contamination, i.e.
