APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1978, p. 121-128 0099-2240/78/0036-0121$02.00/0 Copyright i 1978 American Society for Microbiology
Vol. 36, No. 1 Printed in U.S.A.
Improved Methods for Detecting Enteric Viruses in Oysters MARK D. SOBSEY,* ROBERT J. CARRICK, AND HAROLD R. JENSEN Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, North Carolina 27514 Received for publication 20 December 1977
New and improved methods for concentrating enteroviruses, reoviruses, and adenoviruses from oysters have been developed and evaluated. Viruses are efficiently adsorbed to homogenized oyster meat by adjusting the homogenate to pH 5.0 and a conductivity of -2,000 mg of NaCl per liter. After low-speed centrifugation, the virus-free supernatant is discarded and the viruses are eluted from the sedimented oyster solids with pH 7.5 glycine-NaCl having a conductivity of 8,000 mg of NaCl per liter. The oyster solids are removed by low-speed centrifugation and filtration, and the viruses in the filtered supernatant are concentrated to a small volume by either ultrafiltration or acid precipitation at pH 4.5. The concentrate is treated with antibiotics and inoculated into cell cultures for virus isolation and quantitation. When these methods were tested with oysters experimentally contaminated with polioviruses, reoviruses, and adenoviruses, recovery efficiencies averaged about 46%. With the exception of virus assay and quantitation, these methods are simple and inexpensive enough to be done in typical shellfish microbiology laboratories.
Edible mollusks such as oysters, clams, and mussels can be vehicles for enteric virus disease transmission to humans. Outbreaks of hepatitis A and viral gastroenteritis have been attributed to the consumption of raw and steamed mollusks (1-4, 8, 15, 19). Furthermore, Koff et al. (12) have also suggested that shellfish may be a significant cause of hepatitis A under nonepidemic conditions. The total coliform bacteria standard implemented more than 30 years ago by the National Shellfish Sanitation Program is still used in the United States to determine the microbial quality of shellfish harvesting waters with respect to fecal contamination (24-26), although fecal coliform standards for shellfish and their harvesting waters have more recently been established (22-25). The adequacy of total or fecal coliform standards with respect to enteric virus contamination of shellfish and their harvesting waters has not been directly determined. To be considered entirely adequate as indicator organisms, bacterial indicators such as coliforms must also reflect levels of enteric viruses. However, recent studies by Fugate et al. (7) and Goyal et al. (S. M. Goyal, C. P. Gerba, and J. L. Melnick, J. Water Pollut. Control Fed., in press) suggest that enteric bacterial and viral levels in coastal waters and shellfish may be poorly related. To adequately determine the significance of shellfish as vehicles for enteric virus transmission to humans and the relationship between enteric bacteria and viruses in shellfish, conven-
ient and reliable methods must be developed to quantitatively detect enteric viruses in shellfish. Previous studies on the detection of enteric viruses in oysters, clams, and mussels have used such methods as ethyl ether extraction, fluorocarbon extraction, acid precipitation, adsorption-elution, and ultrafiltration (UF) (9, 10, 13, 14, 17, 18, 21) to concentrate and separate enteric viruses from shellfish tissue components which can interfere with enteric virus detection in cell culture. This study describes the development and evaluation of improved methods for detection of enterovirus, reoviruses, and adenoviruses in oysters that are based on the adsorption-elution-UF method previously developed for enterovirus detection (21). MATERIALS AND METHODS Method of Sobsey et al. (21). In the original
121
method of Sobsey et al. (21) for oysters, enteroviruses are adsorbed to homogenized oyster meats at a pH of 5.5 and a conductivity of c1,500 mg of NaCl per liter. After low-speed centrifugation, the supernatant is discarded and the sedimented virus-containing oyster meat is washed by resuspending in pH 5.5 glycine at low-salt concentration and then centrifuging at low speed. The supernatant is discarded, and the viruses are eluted from the oyster meat by resuspending in pH 3.5 glycine-buffered saline at a conductivity of about 8,000 mg/liter as NaCl. This suspension is centrifuged at low speed, and the virus-containing supernatant is adjusted to pH 7.5 and filtered through a 0.2em-porosity filter. The filtrate is concentrated to a volume of a few milliliters by UF, and the UF concentrate is assayed directly for viruses in cell culture.
122
APPL. ENVIRON. MICROBIOL.
SOBSEY, CARRICK, AND JENSEN
Preliminary studies were conducted with this original ments, shucked oyster meats were drained of excess procedure in order to identify areas for simplification fluid, pooled to give a desired total weight, and experimentally contaminated with viruses as previously deand improvement. Cell cultures. Vero or Buffalo Green Monkey kid- scribed (21). ney (BGMK), continuous cell lines originally derived Filtration and UF of oyster samples. Fiber glass from African green monkey kidneys, were used for (type AP25, Millipore Corp., Bedford, Mass.) and fiber virus growth and assay. Cells were grown in autoclav- glass-asbestos-epoxy (type M-780, series AA, Cox Inable, modified Eagle minimum essential medium with strument Div., Lynch Corp., Detroit, Mich.) filters Earle salts (MEM) supplemented with 10% fetal calf were used in some experiments. UF of samples was done at 4°C in Amicon model 402 stirred pressure cells serum (FCS) and single strength (lx) antibiotics (100 U of penicillin, 100 yg of streptomycin, and 50 ytg of with type PM 30 membranes. kanamycin per ml), and they were maintained in MEM with 2% heat-inactivated FCS and doubleRESULTS strength (2x) antibiotics. Preliminary studies with the original Viruses and virus assays. Representatives from three different enteric virus groups were used in this procedure. Results of preliminary studies with study: poliovirus type 1, strain LSc (enterovirus), reo- the original method indicated that changing the virus type 3, strain Dearing, and the simian adenovirus virus elution pH would be desirable in order to SV-11. Crude virus stocks were prepared by infecting avoid formation of troublesome precipitates that monolayer cultures of Vero (for reovirus and SV-11) frequently developed when the pH 3.5 supernaand BGMK (for poliovirus) cells on maintenance me- tant from the elution step was adjusted to pH dium in 960-cm2 roller bottles at low multiplicity. 7.5. It was also determined that the pH 5.5 When nearly all of the cells developed cytopathic glycine wash procedure would be investigated effects, the cultures were frozen and thawed three to for possible modification or elimination and that five times and clarified by centrifugation at 10,000 x an alternative to the UF concentration proceg and 4°C for 20 min. Adenovirus preparations were concentrated about 10-fold by UF in pressurized, dure would be explored. Virus adsorption to homogenized oysstirred cells using Amicon (Lexington, Mass.) type PM 30 membranes. Crude virus stocks were stored frozen ters. In the previous study by Sobsey et al. (21) at -70°C. Purified, monodispersed virus stocks were two parameters, pH and conductivity, were prepared by Freon extraction and sucrose density gra- found to influence enterovirus adsorption to hodient centrifugation of crude virus as described by mogenized oyster meat. Efficient virus adsorpSharp et al. (20) and Floyd and Sharp (6) and stored tion occurred only at low conductivity (-2,000 at40C. Viruses were assayed by the plaque-forming unit mg of NaCl per liter) and pH values ranging (PFU) method in drained, confluent cultures of Vero from 5.0 to 6.0. Therefore, experiments were done to determine the effect of pH on poliovirus, or BGMK cells grown in either 1-ounce (ca. 30-ml) glass prescription bottles or 30-ml (25-cm2) plastic reovirus, and adenovirus adsorption to homogtissue culture flasks, using 0.1- and 0.5-ml inoculum enized oyster meat at low conductivity. Drained volumes, respectively, and a 1-h adsorption period at oyster meats were blended in enough cold dis370C. If necessary, virus samples were diluted in tilled water so that the homogenate conductivity tris(hydroxymethyl)aminomethane buffered saline was c1,500 mg of NaCl per liter. About 1 x 105 containing 2% FCS and 2x antibiotics. For assaying PFU of test virus was added per ml and the poliovirus and reovirus, a single overlay consisting of homogenate was reblended. Fractions of the viMEM, 1.4% agar (Difco Laboratories, Detroit, Mich.), rus-contaminated homogenate were adjusted to 0.4% NaHCO3, 0.0017% neutral red, 2% heat-inactivated FCS (reovirus only), and either 25 mM MgC12 the pH values shown in Table 1 using either 0.05 (poliovirus) or 0.017% pancreatin (reovirus) was used. Cultures were inverted and incubated at 37°C for TABLE 1. Effect ofpH on virus adsorption to oyster meats at low conductivitya plaque development. For SV-11, a double overlay was used. The first overlay consisted of MEM, 0.9% agar Virus adsorbed (%) (Difco), 0.4% NaHCO3, 2% heat-inactivated FCS, and pH 0.01% diethylaminoethyl-dextran, and cultures were Poliovirus Reovirus Adenovirus incubated at 37°C without inverting. After 5 days, a 3.5 17 13 21 second overlay consisting of MEM, 1.4% agar (Difco), 4.0 NTb 93 81 0.4% NaHCO3, 0.01% diethylaminoethyl-dextran, and 4.5 >99 >99 98 0.003% neutral red was added. Cultures were inverted 5.0 >99 >99 >99 and incubated overnight at 37°C for plaques to become 5.5 >99 >99 98 visible. 6.0 NT 58 99 Oysters and oyster preparation procedures. 6.5 82 73 69 Eastern oysters (Crassostrea virginica) were either 7.5 56 37 44 collected from various growing areas along the North 8.5 19 33 NT Carolina coast or obtained as shell stock from local retailers. In some cases, oysters were shucked and Conductivity,
Vol. 36, No. 1 Printed in U.S.A.
Improved Methods for Detecting Enteric Viruses in Oysters MARK D. SOBSEY,* ROBERT J. CARRICK, AND HAROLD R. JENSEN Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, North Carolina 27514 Received for publication 20 December 1977
New and improved methods for concentrating enteroviruses, reoviruses, and adenoviruses from oysters have been developed and evaluated. Viruses are efficiently adsorbed to homogenized oyster meat by adjusting the homogenate to pH 5.0 and a conductivity of -2,000 mg of NaCl per liter. After low-speed centrifugation, the virus-free supernatant is discarded and the viruses are eluted from the sedimented oyster solids with pH 7.5 glycine-NaCl having a conductivity of 8,000 mg of NaCl per liter. The oyster solids are removed by low-speed centrifugation and filtration, and the viruses in the filtered supernatant are concentrated to a small volume by either ultrafiltration or acid precipitation at pH 4.5. The concentrate is treated with antibiotics and inoculated into cell cultures for virus isolation and quantitation. When these methods were tested with oysters experimentally contaminated with polioviruses, reoviruses, and adenoviruses, recovery efficiencies averaged about 46%. With the exception of virus assay and quantitation, these methods are simple and inexpensive enough to be done in typical shellfish microbiology laboratories.
Edible mollusks such as oysters, clams, and mussels can be vehicles for enteric virus disease transmission to humans. Outbreaks of hepatitis A and viral gastroenteritis have been attributed to the consumption of raw and steamed mollusks (1-4, 8, 15, 19). Furthermore, Koff et al. (12) have also suggested that shellfish may be a significant cause of hepatitis A under nonepidemic conditions. The total coliform bacteria standard implemented more than 30 years ago by the National Shellfish Sanitation Program is still used in the United States to determine the microbial quality of shellfish harvesting waters with respect to fecal contamination (24-26), although fecal coliform standards for shellfish and their harvesting waters have more recently been established (22-25). The adequacy of total or fecal coliform standards with respect to enteric virus contamination of shellfish and their harvesting waters has not been directly determined. To be considered entirely adequate as indicator organisms, bacterial indicators such as coliforms must also reflect levels of enteric viruses. However, recent studies by Fugate et al. (7) and Goyal et al. (S. M. Goyal, C. P. Gerba, and J. L. Melnick, J. Water Pollut. Control Fed., in press) suggest that enteric bacterial and viral levels in coastal waters and shellfish may be poorly related. To adequately determine the significance of shellfish as vehicles for enteric virus transmission to humans and the relationship between enteric bacteria and viruses in shellfish, conven-
ient and reliable methods must be developed to quantitatively detect enteric viruses in shellfish. Previous studies on the detection of enteric viruses in oysters, clams, and mussels have used such methods as ethyl ether extraction, fluorocarbon extraction, acid precipitation, adsorption-elution, and ultrafiltration (UF) (9, 10, 13, 14, 17, 18, 21) to concentrate and separate enteric viruses from shellfish tissue components which can interfere with enteric virus detection in cell culture. This study describes the development and evaluation of improved methods for detection of enterovirus, reoviruses, and adenoviruses in oysters that are based on the adsorption-elution-UF method previously developed for enterovirus detection (21). MATERIALS AND METHODS Method of Sobsey et al. (21). In the original
121
method of Sobsey et al. (21) for oysters, enteroviruses are adsorbed to homogenized oyster meats at a pH of 5.5 and a conductivity of c1,500 mg of NaCl per liter. After low-speed centrifugation, the supernatant is discarded and the sedimented virus-containing oyster meat is washed by resuspending in pH 5.5 glycine at low-salt concentration and then centrifuging at low speed. The supernatant is discarded, and the viruses are eluted from the oyster meat by resuspending in pH 3.5 glycine-buffered saline at a conductivity of about 8,000 mg/liter as NaCl. This suspension is centrifuged at low speed, and the virus-containing supernatant is adjusted to pH 7.5 and filtered through a 0.2em-porosity filter. The filtrate is concentrated to a volume of a few milliliters by UF, and the UF concentrate is assayed directly for viruses in cell culture.
122
APPL. ENVIRON. MICROBIOL.
SOBSEY, CARRICK, AND JENSEN
Preliminary studies were conducted with this original ments, shucked oyster meats were drained of excess procedure in order to identify areas for simplification fluid, pooled to give a desired total weight, and experimentally contaminated with viruses as previously deand improvement. Cell cultures. Vero or Buffalo Green Monkey kid- scribed (21). ney (BGMK), continuous cell lines originally derived Filtration and UF of oyster samples. Fiber glass from African green monkey kidneys, were used for (type AP25, Millipore Corp., Bedford, Mass.) and fiber virus growth and assay. Cells were grown in autoclav- glass-asbestos-epoxy (type M-780, series AA, Cox Inable, modified Eagle minimum essential medium with strument Div., Lynch Corp., Detroit, Mich.) filters Earle salts (MEM) supplemented with 10% fetal calf were used in some experiments. UF of samples was done at 4°C in Amicon model 402 stirred pressure cells serum (FCS) and single strength (lx) antibiotics (100 U of penicillin, 100 yg of streptomycin, and 50 ytg of with type PM 30 membranes. kanamycin per ml), and they were maintained in MEM with 2% heat-inactivated FCS and doubleRESULTS strength (2x) antibiotics. Preliminary studies with the original Viruses and virus assays. Representatives from three different enteric virus groups were used in this procedure. Results of preliminary studies with study: poliovirus type 1, strain LSc (enterovirus), reo- the original method indicated that changing the virus type 3, strain Dearing, and the simian adenovirus virus elution pH would be desirable in order to SV-11. Crude virus stocks were prepared by infecting avoid formation of troublesome precipitates that monolayer cultures of Vero (for reovirus and SV-11) frequently developed when the pH 3.5 supernaand BGMK (for poliovirus) cells on maintenance me- tant from the elution step was adjusted to pH dium in 960-cm2 roller bottles at low multiplicity. 7.5. It was also determined that the pH 5.5 When nearly all of the cells developed cytopathic glycine wash procedure would be investigated effects, the cultures were frozen and thawed three to for possible modification or elimination and that five times and clarified by centrifugation at 10,000 x an alternative to the UF concentration proceg and 4°C for 20 min. Adenovirus preparations were concentrated about 10-fold by UF in pressurized, dure would be explored. Virus adsorption to homogenized oysstirred cells using Amicon (Lexington, Mass.) type PM 30 membranes. Crude virus stocks were stored frozen ters. In the previous study by Sobsey et al. (21) at -70°C. Purified, monodispersed virus stocks were two parameters, pH and conductivity, were prepared by Freon extraction and sucrose density gra- found to influence enterovirus adsorption to hodient centrifugation of crude virus as described by mogenized oyster meat. Efficient virus adsorpSharp et al. (20) and Floyd and Sharp (6) and stored tion occurred only at low conductivity (-2,000 at40C. Viruses were assayed by the plaque-forming unit mg of NaCl per liter) and pH values ranging (PFU) method in drained, confluent cultures of Vero from 5.0 to 6.0. Therefore, experiments were done to determine the effect of pH on poliovirus, or BGMK cells grown in either 1-ounce (ca. 30-ml) glass prescription bottles or 30-ml (25-cm2) plastic reovirus, and adenovirus adsorption to homogtissue culture flasks, using 0.1- and 0.5-ml inoculum enized oyster meat at low conductivity. Drained volumes, respectively, and a 1-h adsorption period at oyster meats were blended in enough cold dis370C. If necessary, virus samples were diluted in tilled water so that the homogenate conductivity tris(hydroxymethyl)aminomethane buffered saline was c1,500 mg of NaCl per liter. About 1 x 105 containing 2% FCS and 2x antibiotics. For assaying PFU of test virus was added per ml and the poliovirus and reovirus, a single overlay consisting of homogenate was reblended. Fractions of the viMEM, 1.4% agar (Difco Laboratories, Detroit, Mich.), rus-contaminated homogenate were adjusted to 0.4% NaHCO3, 0.0017% neutral red, 2% heat-inactivated FCS (reovirus only), and either 25 mM MgC12 the pH values shown in Table 1 using either 0.05 (poliovirus) or 0.017% pancreatin (reovirus) was used. Cultures were inverted and incubated at 37°C for TABLE 1. Effect ofpH on virus adsorption to oyster meats at low conductivitya plaque development. For SV-11, a double overlay was used. The first overlay consisted of MEM, 0.9% agar Virus adsorbed (%) (Difco), 0.4% NaHCO3, 2% heat-inactivated FCS, and pH 0.01% diethylaminoethyl-dextran, and cultures were Poliovirus Reovirus Adenovirus incubated at 37°C without inverting. After 5 days, a 3.5 17 13 21 second overlay consisting of MEM, 1.4% agar (Difco), 4.0 NTb 93 81 0.4% NaHCO3, 0.01% diethylaminoethyl-dextran, and 4.5 >99 >99 98 0.003% neutral red was added. Cultures were inverted 5.0 >99 >99 >99 and incubated overnight at 37°C for plaques to become 5.5 >99 >99 98 visible. 6.0 NT 58 99 Oysters and oyster preparation procedures. 6.5 82 73 69 Eastern oysters (Crassostrea virginica) were either 7.5 56 37 44 collected from various growing areas along the North 8.5 19 33 NT Carolina coast or obtained as shell stock from local retailers. In some cases, oysters were shucked and Conductivity,
