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Differential accumulation and depuration of human enteric viruses by mussels
e
Pergamon
Waf. Sci. Teeh. Vol. 31. No. 5~. pp. 447-451. 1995.
0273-1223(95)OO31O-X
Copyright C 1995 IAWQ Printed in Great Britaio. All rigbll reserved. 0273-12.23195 $9·S0 + 0-00
DIFFERENTIAL ACCUMULATION AND DEPURATION OF HUMAN ENTERIC VIRUSES BY MUSSELS A. Bosch, R. M. Pint6 and F. X. Abad Department ofMicrobiology, University ofBarcelona. 08028 Barcelona, Spain
ABSTRACT The tissue distribution of adenovirus 40 (ADV) and human rotavirus, serotype 3 (HRV) was determined after feeding the CODUJIQD mussel (Mytilus spp.) with high levels of clay-associated virus. At different time interVals, individual tissues were carefully dissected and assayed for infectivity. Viruses were detected in contaminated mussels after I-hour contact, and maximum levels were observed after 6 hours. Most infectious viruses were located in the gills and in the digestive tract. Decreasing virus numbers were found in the mantle lobes. Mussels contaminated with poliovirus I (PV), hepatitis A virus. strain HM-175 (HAY), ADV. HRV. and bacteriophages of Bacteroides fragilis (840-8) were depurated in SO-I tanks with a continuous now of Olonated marine water. Arter 96 hours. HAV and HRV suffered less than 2 Log 10 titre reduction (LTR). while ADV showed a 2.7 LTR. PV showed a 3 LTR after 48 hours and became undetectable thereafter. Bacteriophage 840-8 suffered less than 2 LTR after 96 hours, suggesting that it could be an appropriate indicator of the efficiency of virus elimination during shellfISh depuration.
KEYWORDS
Mussels; human enteric viruses; uptake; depuration; bacteriophages.
INIRODUcnON Human pathogenic viruses enter the marine environment primarily through the discharge of treated and untreated sewage into surface waters, since current treatment practices do not provide virus-free effluents (Rao and Melnick, 1986). The ability of shellfish to accumulate microorganisms during feeding is well documented (Gerbu, 1988). Shellfish grown and harvested from sewage polluted waters have been implicated in outbreaks of viral diseases. Although most data available on the bioaccumulation and depuration of viruses in shellfish have been obtained investigating enteroviruses (Di Girolamo et aI.• 1975; Metcalf et at., 1980), frequent outbreaks of shellfish-borne viral gastroenteritis have been reported (Richards. 1987; Hu et at., 1991). Depuration of bivalves exploits the feeding physiology of shellfish as a means of reversing contaminant ingestion. Shellfish are placed in clean seawater to allow them to purge themselves of their microbial load. The effectiveness of this procedure for the removal of viruses is variable (Gerba, 1988). This study was undertaken to determine the tissue distribution of enteric adenovirus 40 and human rotavirus after feeding the mussels with high levels of clay-associated virus. It was also designed to obtain 447
A. BOSCH tt al.
448
experimental evidence of the effect of depuration on the removal of poliovirus, hepatitis A virus, adenovirus rotavirus and bacteriophages of Bacteroides fragilis. ' MATERIALS AND METHODS
Viruses and cell cultures. Poliovirus I, strain LSc 2ab (PY), and human rotavirus Ito r p13 (HRV) were propagated and assayed in BGM and MA-I04 cells, respectively, as previously described (Bosch et al., 1991). FRhK-4 cell cultures were used to propagate and assay the cytopathogenic HM-175 (courtesy ofT. Cromeans, Centres for Disease Control, Atlanta, Ga) strain of hepatitis A virus (HAY) (Cromeans et al., 1987). Human enteric adenovirus type 40 (ADY, courtesy of W.O.K. Grabow, University of Pretoria, South Africa) was cultivated and assayed in CaCo-2 cell monolayers (Pint6 et al., 1994). Bacteriophage B40-8 of Bacteroides fragilis was assayed as previously described (Tartera et ai., 1992). Experimental contamination of mussels. Five groups of forty specimens of the common mussel, Mytilus spp., were contaminated over a 24 hour period with approximately 107-10 8 MPNCU of clay-associated viruses, in 4-litre tanks of estuarine water (3.2% salinity, conductivity <3500 IJ.Illhos, 21-23OC temperature). Mussels were kept in starvation for 24 h before each experiment Mussel depuration. Depuration was perfonned over 96 hours by placing groups of 40 experimentally contaminated mussels in a continuous flow of ozonated marine water in 50 I tanks. Assay procedures. At designated time intervals. viruses were extracted from mussel samples as described elsewhere (Sobsey et al., 1978) and concentrated from the resulting eluate by polyethylenglycol precipitation (Lewis and Metcalf, 1988). In the bioaccumulation experiments, whole mussel meat and dissected tissues (gills, digestive tract and mantle lobes) were assayed for viruses. Only whole mussel meat was processed in the depuration experiments. All experiments were conducted at least in duplicate, and all virus assays were perfonned twice.
ADV
HRV
175000
12000
u
150000
10000
::2
125000
8000
rn t>J rn
100000
ao ........ :::::>
Z ll.
'-'
:::>
~
75000
Q
50000
;; t>J a:l ~
0
rn
4000 2000
25000
Q
0
<
" o
~
6000
INTERV.
0 3 6 9 12 15 18 21 24
HOURS
0
GILL
HOURS 6
MANTLE
'V
DIGEST
•
TOTAL
Figure J. ERleric adenoviruses (ADY) and buman rotavirusess (HRV) adsorbed to mussel tissues.
Differential accumulation and depuration of human enteric viruses
449
RESULTS
Bioaccumulation of viruses in mussels. Mter feeding mussels with high levels of clay-associated ADV and HRV, infectious viruses were detected in contaminated mussels after I hour contact time (Fig. I). Maximum levels of viruses adsorbed to mussel meat were observed after 6 hours: 1.7xlOS MPNCU/g for ADV, and l.lxl()4 MPNCUlg for HRV. Infectious virus titres declined thereafter. The water housing the mussels was monitored for the presence of infectious viruses throughout the experiment. ADV levels per litre of water were 2.5x107, 1.7xl07 and 4.0x106 MPNCU at 0, 6 and 24 hour time, respectively. At the same sampling times, HRV levels were 8.0x106, 5.0x106 and 8.0dO S MPNCUIl. ADV and HRV adsorbed to mussel tissue after 6 hours represent 25% and 35% of the total seeded viruses, respectively. In another set of experiments, 56% of HAV, 4% of PV and 5% of 840-8 were adsorbed to mussels after 6 hours. Tissue distribution of viruses in mussels. Dissected tissues and intervalvar fluid from experimentally contaminated mussels were assayed for infectious ADV and HRV (Fig. I). Maximum levels of ADV were detected in the intervalvar fluid, followed by gills. digestive tract and mantle lobes. For HRV. the highest virus numbers were found in gills and labial palps, followed by digestive tract, intervalvar fluid and mantle lobes. After 6 hours, the percent tissue distribution of adsorbed adenoviruses was 20% in gills, 5% in the mantle lobes and 12% in the digestive tract, while 27% was detected in the intervalvar fluid. For HRV, after the same contact time. these figures were 49% in gills. 4% in mantle lobes, 27% in digestive tract and 22% in intervalvar fluid. Elimination of viruses by depuration. The effects of depuration on the removal of PV. HAV. ADV, HRV, and bacteriophage B40-8 from mussel tissue are depicted in Fig. 2. Experimentally contaminated mussels were kept for 96 h in 50-1 tanks with a continuous flow of ozonated marine water. The most dramatic effect of depuration was on PV, which showed a 3 10glO titre reduction (LTR) after 48 hours, and became undetectable thereafter. In contrast, after 96 hours, HAV and HRV suffered less than 2 L TR, while ADV showed a 2.7 LTR. Less than 2 LTR of the levels of bacteriophage B40-8 was observed after 96 hours of depuration.
a
....o
-2
._-._--.
ljlJ
o
...J
-3
_4'--_...-J'--_--1_ _- - L_ _
o
24
4P,
72
•
PV
•
ADV
A
HAV
'f'
HRV
o
840-8
~
96
TlME (Hours)
Figure 2. Elimination of viruses by mussel depuration.
A. BOSCH et al.
450
DISCUSSION The bioaccumulation of viruses in mussels is function of the physiology of bivalves. Under optimal conditions, mussels are reported to fIlter between 0.2 and 5 litres of water per hour (J. Amengual, personal communication). It has been shown that the presence of particulate material enhances virus uPtake by shellfish (Metcalf et al., 1980). In these experiments, 4-56% of clay-associated viruses present in the Water were adsorbed to mussel tissue. In some studies of artificial contamination of shellfIsh in flowing seawater systems, the majority of viruses were sequestered within the digestive tract (Metcalf et al.• 1980). In this work, the tissue localization of AnV differed from that of HRV. It seems reasonable to believe that differences in the virus strain used and in contact times could account for some discordances found in the literature on the localization of viruses adsorbed to shellfish (Di Girolamo et al., 1975; Power and Collins, 1990). The effIciency of virus uptake and removal from mussel does not seem to be only a matter of the size of the virus particle. Two 27-nm viruses, PV and HAV were differentially adsorbed and depurated in our studies, and AnV were more efficiently removed than other viruses of related size such as HRV. It has been stated that virus contamination of shellfIsh is transient (Power and Collins. 1990). However, it is clear that under the depuration conditions used in these studies. periods of depuration longer than 36 hours. as established in the current standards (Boletfn OfIcial del Estado, 1985), are required for the total elimination of viral pathogens. Currently. the regulation of shellfIsh and their growing waters is based on bacterial standards (U.S. Public Health Service, 1965; Boletfn OfIcial del Estado. 1985), and most studies on virus adsorption and removal from shellfIsh have been performed with poliovirus. However, hepatitis A cases have been associated with the consumption of shellfIsh controlled through public health measures (Mele et aI., 1989), and human rotaviruses and hepatitis A virus have been detected in mussels that met bacteriological criteria, and thus were legally adequate for public consumption (Jofre et al.• 1993). In this work HRV, HAV and AnV persisted much longer than PV under commercial depuration conditions. An avenue that must be pursued to reduce the threat of shellfIsh-borne disease outbreaks is to correlate the presence of indicator microorganisms with human pathogenic viruses. in an effort to determine their adequacy as indicators of viral contamination. In previous studies, bacteriophages of B. jragilis have been shown to be promising indicators of the virological quality of water (fartera et al., 1987). In this work. the inactivation of bacteriophage B40-8 after a 96-hour mussel depuration treatment was similar to that of HRV or HAV. Under the same conditions, AnV and particularly PV infectivity was more efficiently removed. These data suggest that B. fragilis phages could be appropriate and reliable indicators of the effIciency of virus elimination during shellfIsh depuration. Improved monitoring of shellfIsh growing areas and enforcement of applicable laws and regulations governing the harvesting, processing and distribution of shellfIsh should be developed. If bacterial indicators are unacceptable as indicators of viral pathogens (Bosch et al., 1991), guidelines restricting the levels of enteric viruses themselves or reliable alternative bacteriophage indicators in shellfIsh and harvest waters should be implemented. ACKNOWLEDGEMENTS
F.x. Abad was recipient of a F.I. fellowship from
the Generalitat de Catalunya. We are grateful to Dr. J. Amengual for providing mussels and for useful discussion. REFERENCES
Boletin Oficial del Estado. (1985). DecrelD de 31 de Mayo de 1985 B.O.E. 137. 17298-17300. Bosch, A•• Lucena, F. Diez, I.M., Gajardo, Ro, Blasi, M and lofre, I. (1991). Human enteric viruses and indicator microorganisms in a water supply associated with an outbreak of infectious hepatitis. J. Amer. Water Works Assoc. 83, 80-83.
Differential accumulation and depuration of buman enteric viruses
4S1
Cromeans, T•• Sobsey. M.D•• Fields, H.A. (1987): Development of a plaque assay for a cytopathic rapidly replicating isolate of bepatitis A virus. J. Med. ViroL 22, 4S-S6. Di Girolamo, R.. Liston, J. and Matcbes, J. (l97S). Uptake and elimination of poliovirus by West coast oysters. AppL MicrobioL 29. 260-264.
Gerba, C.P (1988). Viral disease transmission by seafoods. Food TechnoL 42,99-103. Hu, M., Kang, L. and Yao, G. (l99\). An outbreak of viral hepatitis in Shanghai. /n L. Bianchi, W. Gerok, K.P. Maier and F. Deinbardt (Ells.) Infectious diseases of the liver, Falk symposium 54, pp 361-372. Jofre, J.• Lucena. F., Gajardo, R. and Boscb, A. (1993). Detection of human enteric viruses in mussels (Mytilus edulis). /n S.R. Farrah, C.P. Gerba & R. Walter (Ells) Contamination of the environment by viruses and methods of control, Wiener Mitteilungen, Band 112, pp IOS-1I0. Lewis, G.D. and Metcalf. T.G. (1988). Polyethylene glycol precipitation for recovery of pathogenic viruses. including hepatitis A virus and buman rotavirus, from oyster, water, and sediment samples. AppL Environ. MicrobioL 54, 1983-1988. Metca1f, T.G.• Eckerson, D.• Moulton, E. and Larkin, E.P. (1980). Uptake and depletion of particulate-associated poliovirus by the soft shell clam. J. Food Protec. 43, 87-88. Mele, A.• Rastelli, M.G., Gill., O.N., DiBisceglie, D., Rosmini, F., Pardelli, G., Valtriani, C. and Patriarcbi, P. (1989). Recurrent epidemic hepatitis A associated with consumption of raw sbellflsh. probably controlled through public bealth measures. Amer. J. EpidemioL 130. 540-S46. Pint6, R.M•• Diez, J.M and Bosch. A. (\994). Use of the colonic carcinoma cell line CaCo-2 for the in vivo amplification and detection of enteric viruses. J. Med. ViraL (In Press). power. UF. and Collins, J.K. (\990). Tissue distribution of a colipbage and Escherichia coli in mussels after contamination and depuration.AppL Environ. Microbial. 56, 803-807. Rao. V.C. and Melnick, J.L.. (1986). Environmental Virology. /n J.A. Cole, C,J.Knowles. D. ScWessinger (Ells) Aspects of Microbiology 13. American Society for Microbiology, Washington, D.C. Richards. G.P. (1987). Shellfish-associated enteric virus illness in the United States, 1934-1984. Estuaries 10, 84-8S. Sobsey, M.D., Carrick. R.J. and Jensen, H.R. (1978). Improved methods for detecting enteric viruses in oysters. AppL Environ. MicrobioL,36, 121-128. Tartera, C. and J. Jofre. 1987. Bacteriophages active against Bacteroides fragilis in sewage-polluted waters. AppL Environ. MicrobioL 53,1632-1637. Tartera, C., Araujo, R., Michel, T~ and Jofre, J. (1992). Culture and decontamination methods affecting enumeration of phages infecting Bacteroides fragilis in sewage. Appl. Environ. MicrobioL 58, 2670-2673. United States Public Health Service (196S). National shellfish sanitation program manual of operations. Part I, Sanitation of shellfJsh growing areas. Publ. No. 33. Washington, D.C.
Pergamon
Waf. Sci. Teeh. Vol. 31. No. 5~. pp. 447-451. 1995.
0273-1223(95)OO31O-X
Copyright C 1995 IAWQ Printed in Great Britaio. All rigbll reserved. 0273-12.23195 $9·S0 + 0-00
DIFFERENTIAL ACCUMULATION AND DEPURATION OF HUMAN ENTERIC VIRUSES BY MUSSELS A. Bosch, R. M. Pint6 and F. X. Abad Department ofMicrobiology, University ofBarcelona. 08028 Barcelona, Spain
ABSTRACT The tissue distribution of adenovirus 40 (ADV) and human rotavirus, serotype 3 (HRV) was determined after feeding the CODUJIQD mussel (Mytilus spp.) with high levels of clay-associated virus. At different time interVals, individual tissues were carefully dissected and assayed for infectivity. Viruses were detected in contaminated mussels after I-hour contact, and maximum levels were observed after 6 hours. Most infectious viruses were located in the gills and in the digestive tract. Decreasing virus numbers were found in the mantle lobes. Mussels contaminated with poliovirus I (PV), hepatitis A virus. strain HM-175 (HAY), ADV. HRV. and bacteriophages of Bacteroides fragilis (840-8) were depurated in SO-I tanks with a continuous now of Olonated marine water. Arter 96 hours. HAV and HRV suffered less than 2 Log 10 titre reduction (LTR). while ADV showed a 2.7 LTR. PV showed a 3 LTR after 48 hours and became undetectable thereafter. Bacteriophage 840-8 suffered less than 2 LTR after 96 hours, suggesting that it could be an appropriate indicator of the efficiency of virus elimination during shellfISh depuration.
KEYWORDS
Mussels; human enteric viruses; uptake; depuration; bacteriophages.
INIRODUcnON Human pathogenic viruses enter the marine environment primarily through the discharge of treated and untreated sewage into surface waters, since current treatment practices do not provide virus-free effluents (Rao and Melnick, 1986). The ability of shellfish to accumulate microorganisms during feeding is well documented (Gerbu, 1988). Shellfish grown and harvested from sewage polluted waters have been implicated in outbreaks of viral diseases. Although most data available on the bioaccumulation and depuration of viruses in shellfish have been obtained investigating enteroviruses (Di Girolamo et aI.• 1975; Metcalf et at., 1980), frequent outbreaks of shellfish-borne viral gastroenteritis have been reported (Richards. 1987; Hu et at., 1991). Depuration of bivalves exploits the feeding physiology of shellfish as a means of reversing contaminant ingestion. Shellfish are placed in clean seawater to allow them to purge themselves of their microbial load. The effectiveness of this procedure for the removal of viruses is variable (Gerba, 1988). This study was undertaken to determine the tissue distribution of enteric adenovirus 40 and human rotavirus after feeding the mussels with high levels of clay-associated virus. It was also designed to obtain 447
A. BOSCH tt al.
448
experimental evidence of the effect of depuration on the removal of poliovirus, hepatitis A virus, adenovirus rotavirus and bacteriophages of Bacteroides fragilis. ' MATERIALS AND METHODS
Viruses and cell cultures. Poliovirus I, strain LSc 2ab (PY), and human rotavirus Ito r p13 (HRV) were propagated and assayed in BGM and MA-I04 cells, respectively, as previously described (Bosch et al., 1991). FRhK-4 cell cultures were used to propagate and assay the cytopathogenic HM-175 (courtesy ofT. Cromeans, Centres for Disease Control, Atlanta, Ga) strain of hepatitis A virus (HAY) (Cromeans et al., 1987). Human enteric adenovirus type 40 (ADY, courtesy of W.O.K. Grabow, University of Pretoria, South Africa) was cultivated and assayed in CaCo-2 cell monolayers (Pint6 et al., 1994). Bacteriophage B40-8 of Bacteroides fragilis was assayed as previously described (Tartera et ai., 1992). Experimental contamination of mussels. Five groups of forty specimens of the common mussel, Mytilus spp., were contaminated over a 24 hour period with approximately 107-10 8 MPNCU of clay-associated viruses, in 4-litre tanks of estuarine water (3.2% salinity, conductivity <3500 IJ.Illhos, 21-23OC temperature). Mussels were kept in starvation for 24 h before each experiment Mussel depuration. Depuration was perfonned over 96 hours by placing groups of 40 experimentally contaminated mussels in a continuous flow of ozonated marine water in 50 I tanks. Assay procedures. At designated time intervals. viruses were extracted from mussel samples as described elsewhere (Sobsey et al., 1978) and concentrated from the resulting eluate by polyethylenglycol precipitation (Lewis and Metcalf, 1988). In the bioaccumulation experiments, whole mussel meat and dissected tissues (gills, digestive tract and mantle lobes) were assayed for viruses. Only whole mussel meat was processed in the depuration experiments. All experiments were conducted at least in duplicate, and all virus assays were perfonned twice.
ADV
HRV
175000
12000
u
150000
10000
::2
125000
8000
rn t>J rn
100000
ao ........ :::::>
Z ll.
'-'
:::>
~
75000
Q
50000
;; t>J a:l ~
0
rn
4000 2000
25000
Q
0
<
" o
~
6000
INTERV.
0 3 6 9 12 15 18 21 24
HOURS
0
GILL
HOURS 6
MANTLE
'V
DIGEST
•
TOTAL
Figure J. ERleric adenoviruses (ADY) and buman rotavirusess (HRV) adsorbed to mussel tissues.
Differential accumulation and depuration of human enteric viruses
449
RESULTS
Bioaccumulation of viruses in mussels. Mter feeding mussels with high levels of clay-associated ADV and HRV, infectious viruses were detected in contaminated mussels after I hour contact time (Fig. I). Maximum levels of viruses adsorbed to mussel meat were observed after 6 hours: 1.7xlOS MPNCU/g for ADV, and l.lxl()4 MPNCUlg for HRV. Infectious virus titres declined thereafter. The water housing the mussels was monitored for the presence of infectious viruses throughout the experiment. ADV levels per litre of water were 2.5x107, 1.7xl07 and 4.0x106 MPNCU at 0, 6 and 24 hour time, respectively. At the same sampling times, HRV levels were 8.0x106, 5.0x106 and 8.0dO S MPNCUIl. ADV and HRV adsorbed to mussel tissue after 6 hours represent 25% and 35% of the total seeded viruses, respectively. In another set of experiments, 56% of HAV, 4% of PV and 5% of 840-8 were adsorbed to mussels after 6 hours. Tissue distribution of viruses in mussels. Dissected tissues and intervalvar fluid from experimentally contaminated mussels were assayed for infectious ADV and HRV (Fig. I). Maximum levels of ADV were detected in the intervalvar fluid, followed by gills. digestive tract and mantle lobes. For HRV. the highest virus numbers were found in gills and labial palps, followed by digestive tract, intervalvar fluid and mantle lobes. After 6 hours, the percent tissue distribution of adsorbed adenoviruses was 20% in gills, 5% in the mantle lobes and 12% in the digestive tract, while 27% was detected in the intervalvar fluid. For HRV, after the same contact time. these figures were 49% in gills. 4% in mantle lobes, 27% in digestive tract and 22% in intervalvar fluid. Elimination of viruses by depuration. The effects of depuration on the removal of PV. HAV. ADV, HRV, and bacteriophage B40-8 from mussel tissue are depicted in Fig. 2. Experimentally contaminated mussels were kept for 96 h in 50-1 tanks with a continuous flow of ozonated marine water. The most dramatic effect of depuration was on PV, which showed a 3 10glO titre reduction (LTR) after 48 hours, and became undetectable thereafter. In contrast, after 96 hours, HAV and HRV suffered less than 2 L TR, while ADV showed a 2.7 LTR. Less than 2 LTR of the levels of bacteriophage B40-8 was observed after 96 hours of depuration.
a
....o
-2
._-._--.
ljlJ
o
...J
-3
_4'--_...-J'--_--1_ _- - L_ _
o
24
4P,
72
•
PV
•
ADV
A
HAV
'f'
HRV
o
840-8
~
96
TlME (Hours)
Figure 2. Elimination of viruses by mussel depuration.
A. BOSCH et al.
450
DISCUSSION The bioaccumulation of viruses in mussels is function of the physiology of bivalves. Under optimal conditions, mussels are reported to fIlter between 0.2 and 5 litres of water per hour (J. Amengual, personal communication). It has been shown that the presence of particulate material enhances virus uPtake by shellfish (Metcalf et al., 1980). In these experiments, 4-56% of clay-associated viruses present in the Water were adsorbed to mussel tissue. In some studies of artificial contamination of shellfIsh in flowing seawater systems, the majority of viruses were sequestered within the digestive tract (Metcalf et al.• 1980). In this work, the tissue localization of AnV differed from that of HRV. It seems reasonable to believe that differences in the virus strain used and in contact times could account for some discordances found in the literature on the localization of viruses adsorbed to shellfish (Di Girolamo et al., 1975; Power and Collins, 1990). The effIciency of virus uptake and removal from mussel does not seem to be only a matter of the size of the virus particle. Two 27-nm viruses, PV and HAV were differentially adsorbed and depurated in our studies, and AnV were more efficiently removed than other viruses of related size such as HRV. It has been stated that virus contamination of shellfIsh is transient (Power and Collins. 1990). However, it is clear that under the depuration conditions used in these studies. periods of depuration longer than 36 hours. as established in the current standards (Boletfn OfIcial del Estado, 1985), are required for the total elimination of viral pathogens. Currently. the regulation of shellfIsh and their growing waters is based on bacterial standards (U.S. Public Health Service, 1965; Boletfn OfIcial del Estado. 1985), and most studies on virus adsorption and removal from shellfIsh have been performed with poliovirus. However, hepatitis A cases have been associated with the consumption of shellfIsh controlled through public health measures (Mele et aI., 1989), and human rotaviruses and hepatitis A virus have been detected in mussels that met bacteriological criteria, and thus were legally adequate for public consumption (Jofre et al.• 1993). In this work HRV, HAV and AnV persisted much longer than PV under commercial depuration conditions. An avenue that must be pursued to reduce the threat of shellfIsh-borne disease outbreaks is to correlate the presence of indicator microorganisms with human pathogenic viruses. in an effort to determine their adequacy as indicators of viral contamination. In previous studies, bacteriophages of B. jragilis have been shown to be promising indicators of the virological quality of water (fartera et al., 1987). In this work. the inactivation of bacteriophage B40-8 after a 96-hour mussel depuration treatment was similar to that of HRV or HAV. Under the same conditions, AnV and particularly PV infectivity was more efficiently removed. These data suggest that B. fragilis phages could be appropriate and reliable indicators of the effIciency of virus elimination during shellfIsh depuration. Improved monitoring of shellfIsh growing areas and enforcement of applicable laws and regulations governing the harvesting, processing and distribution of shellfIsh should be developed. If bacterial indicators are unacceptable as indicators of viral pathogens (Bosch et al., 1991), guidelines restricting the levels of enteric viruses themselves or reliable alternative bacteriophage indicators in shellfIsh and harvest waters should be implemented. ACKNOWLEDGEMENTS
F.x. Abad was recipient of a F.I. fellowship from
the Generalitat de Catalunya. We are grateful to Dr. J. Amengual for providing mussels and for useful discussion. REFERENCES
Boletin Oficial del Estado. (1985). DecrelD de 31 de Mayo de 1985 B.O.E. 137. 17298-17300. Bosch, A•• Lucena, F. Diez, I.M., Gajardo, Ro, Blasi, M and lofre, I. (1991). Human enteric viruses and indicator microorganisms in a water supply associated with an outbreak of infectious hepatitis. J. Amer. Water Works Assoc. 83, 80-83.
Differential accumulation and depuration of buman enteric viruses
4S1
Cromeans, T•• Sobsey. M.D•• Fields, H.A. (1987): Development of a plaque assay for a cytopathic rapidly replicating isolate of bepatitis A virus. J. Med. ViroL 22, 4S-S6. Di Girolamo, R.. Liston, J. and Matcbes, J. (l97S). Uptake and elimination of poliovirus by West coast oysters. AppL MicrobioL 29. 260-264.
Gerba, C.P (1988). Viral disease transmission by seafoods. Food TechnoL 42,99-103. Hu, M., Kang, L. and Yao, G. (l99\). An outbreak of viral hepatitis in Shanghai. /n L. Bianchi, W. Gerok, K.P. Maier and F. Deinbardt (Ells.) Infectious diseases of the liver, Falk symposium 54, pp 361-372. Jofre, J.• Lucena. F., Gajardo, R. and Boscb, A. (1993). Detection of human enteric viruses in mussels (Mytilus edulis). /n S.R. Farrah, C.P. Gerba & R. Walter (Ells) Contamination of the environment by viruses and methods of control, Wiener Mitteilungen, Band 112, pp IOS-1I0. Lewis, G.D. and Metcalf. T.G. (1988). Polyethylene glycol precipitation for recovery of pathogenic viruses. including hepatitis A virus and buman rotavirus, from oyster, water, and sediment samples. AppL Environ. MicrobioL 54, 1983-1988. Metca1f, T.G.• Eckerson, D.• Moulton, E. and Larkin, E.P. (1980). Uptake and depletion of particulate-associated poliovirus by the soft shell clam. J. Food Protec. 43, 87-88. Mele, A.• Rastelli, M.G., Gill., O.N., DiBisceglie, D., Rosmini, F., Pardelli, G., Valtriani, C. and Patriarcbi, P. (1989). Recurrent epidemic hepatitis A associated with consumption of raw sbellflsh. probably controlled through public bealth measures. Amer. J. EpidemioL 130. 540-S46. Pint6, R.M•• Diez, J.M and Bosch. A. (\994). Use of the colonic carcinoma cell line CaCo-2 for the in vivo amplification and detection of enteric viruses. J. Med. ViraL (In Press). power. UF. and Collins, J.K. (\990). Tissue distribution of a colipbage and Escherichia coli in mussels after contamination and depuration.AppL Environ. Microbial. 56, 803-807. Rao. V.C. and Melnick, J.L.. (1986). Environmental Virology. /n J.A. Cole, C,J.Knowles. D. ScWessinger (Ells) Aspects of Microbiology 13. American Society for Microbiology, Washington, D.C. Richards. G.P. (1987). Shellfish-associated enteric virus illness in the United States, 1934-1984. Estuaries 10, 84-8S. Sobsey, M.D., Carrick. R.J. and Jensen, H.R. (1978). Improved methods for detecting enteric viruses in oysters. AppL Environ. MicrobioL,36, 121-128. Tartera, C. and J. Jofre. 1987. Bacteriophages active against Bacteroides fragilis in sewage-polluted waters. AppL Environ. MicrobioL 53,1632-1637. Tartera, C., Araujo, R., Michel, T~ and Jofre, J. (1992). Culture and decontamination methods affecting enumeration of phages infecting Bacteroides fragilis in sewage. Appl. Environ. MicrobioL 58, 2670-2673. United States Public Health Service (196S). National shellfish sanitation program manual of operations. Part I, Sanitation of shellfJsh growing areas. Publ. No. 33. Washington, D.C.