- Email: [email protected]
Radiation sensitivity of poliovirus, a model for norovirus, inoculated in oyster (Crassostrea gigas) and culture broth under different conditions
ARTICLE IN PRESS Radiation Physics and Chemistry 78 (2009) 597–599
Contents lists available at ScienceDirect
Radiation Physics and Chemistry journal homepage: www.elsevier.com/locate/radphyschem
Radiation sensitivity of poliovirus, a model for norovirus, inoculated in oyster (Crassostrea gigas) and culture broth under different conditions Pil-Mun Jung a, Jae Seok Park b, Jin-Gyu Park a, Jae-Nam Park a, In-Jun Han a, Beom-Seok Song a, Jong-il Choi a, Jae-Hun Kim a, Myung-Woo Byun a, Min Baek c, Young-Jin Chung d, Ju-Woon Lee a, a
Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185, South Korea Korea Food & Drug Administration, Seoul 122-704, South Korea c Atomic Energy Policy Division, Ministry of Education, Science and Technology, Gwacheon 427-715, South Korea d Department of Food and Nutrition, Chungnam National University, Daejeon 305-764, South Korea b
a r t i c l e in fo
Keywords: Radiation sensitivity Poliovirus Oyster Gamma irradiation
abstract Poliovirus is a recognized surrogate for norovirus, pathogen in water and food, due to the structural and genetic similarity. Although radiation sensitivity of poliovirus in water or media had been reported, there has been no research in food model such as shellfish. In this study, oyster (Crassostrea gigas) was incubated in artificial seawater contaminated with poliovirus, and thus radiation sensitivity of poliovirus was determined in inoculated oyster. The effects of ionizing radiation on the sensitivity of poliovirus were also evaluated under different conditions such as pH (4–7) and salt concentration (1–15%) in culture broth, and temperature during irradiation. The D10 value of poliovirus in PBS buffer, virus culture broth and oyster was determined to 0.46, 2.84 and 2.94 kGy, respectively. The initial plaque forming unit (PFU) of poliovirus in culture broth was slightly decreased as the decrease of pH and the increase of salt concentration, but radiation sensitivity was not affected by pH and salt contents. However, radiation resistance of poliovirus was increased at frozen state. These results provide the basic information for the inactivation of pathogenic virus in foods by using irradiation. & 2009 Published by Elsevier Ltd.
1. Introduction Despite the introduction of sanitary controls regulating their production, shellfish, particularly bivalve molluscs, continue to feature highly in statistics of food-borne disease (Potasman et al., 2002; Sumner and Ross, 2002). Recent decades have also seen a significant change in the reported etiology of shellfish-borne illness. Epidemiological evidence suggests that human enteric viruses, notably norovirus, formerly ‘norwalk-like virus’ or small round structured viruses, and hepatitis A virus (HAV), are now the most common cause of shellfish-borne illness (Gillespie et al., 2001; Lees, 2000). These changes may be explained, in part, by the increased surveillance for these viruses and recognition of their importance in terms of human health, but it is also likely to reflect limitations in the current production and purification processes used for shellfish. Further processing may, therefore, be required to improve the safety of shellfish for human consumption. However, shellfish such as oysters and clams are often eaten raw or lightly cooked and this not only has implications for disease transmission, but also limits processing methods suitable
for these products. The preservation methods, such as highpressure and thermal processing, can be used to eliminate a pathogenic virus (Slomka and Appleton, 1998). However, these processes will not only alter the sensory and nutritional qualities of the food, but convert the product into cooked oysters. Irradiation treatment is being used more frequently as a useful and effective means of disinfection without compromising sensory and nutritional quality (Sommer et al., 2001). Poliovirus is recognized as a surrogate for norovirus used in inactivation studies, because norovirus cannot be cultured (Duizer et al., 2004). Although radiation sensitivity of poliovirus in water or media had been reported (Husman et al., 2004), there was no research in food model such as shellfish. Therefore, the aim of this study was to determine the radiation sensitivity of poliovirus inoculated in oyster and evaluate the effect of ionizing radiation on the sensitivity of poliovirus under different conditions.
2. Materials and methods 2.1. Cell and virus culture
Corresponding author. Tel.: +82 63 570 3204; fax: +82 63 570 3207.
E-mail address: [email protected] (J.-W. Lee). 0969-806X/$ - see front matter & 2009 Published by Elsevier Ltd. doi:10.1016/j.radphyschem.2009.03.017
LLC-MK2 (ATCC number: CCL-7.1TM) cell was grown in 75 cm2 plastic cell culture flasks in Eagle’s minimal essential medium
ARTICLE IN PRESS 598
P.-J. Jung et al. / Radiation Physics and Chemistry 78 (2009) 597–599
(MEM) supplemented with 10% irradiated fetal bovine serum (FBS). Poliovirus, serotype poliovirus-1 (ATCC VR-1562TM) was propagated in LLC-MK2 monolayer. Flask monolayer cultures were inoculated with LLC-MK2 cell incubated until a visible cytopathic effect (CPE) affecting approximately 80% of the cells was observed. The cultures were then frozen and thawed three times, centrifuged at 1000g for 20 min to remove cell debris, and the supernatants pooled and stored at 70 1C prior to use.
Table 1 Radiosensitivity (D10) of poliovirus in various irradiation conditions. Irradiation condition
D10 value (kGy)
Oyster Culture broth PBS buffer pH
7.0 6.0 5.0 4.0
2.84 2.94 0.46 3.71 3.40 3.73 3.39
Salt concentration (%)
1.0 3.5 7.0 15.0
2.94 3.01 3.11 3.14
Irradiation temperature (1C)
Room temp. Refrigeration on ice Freezing on dry ice
3.39 3.22 8.25
2.2. Shellfish maintenance and inoculation of poliovirus Artificial seawater was prepared according to the method described by Kester and Pytkowicz (1967) and this artificial seawater was then added to commercial depuration tank. The depuration system was run overnight to aerate the water and stabilize the temperature at 17 1C. Oysters (Crassostrea gigas) were obtained from Tongyeong Oyster Fishery, Tongyeong City, Gyeongsangnam-do, Korea. The oysters were washed to remove debris, placed in plastic breadbaskets, and transferred to the depuration tank for 48 h prior to use. The natural accumulation of viruses by shellfish was simulated by incubating batches of oysters at 16 1C in the dark for 2 h in 7 L artificial seawater to which was added 50 ml poliovirus stock with an infectivity titer of 8.0870.15 TCID50 (tissue culture infectious dose, log10). This resulted in seawater containing 4.2370.33 TCID50 of poliovirus. Air was pumped via an aquarium pump into the seawater to ensure adequate aeration throughout the inoculation period. 2.3. Sample preparation and gamma irradiation To adjust pH of salt concentration in culture broth, 5 M HCl and 1 M NaOH and NaCl were used to make virus suspension in MEM covering the pH range from 4 to 7 and the salt concentration range from 1% to 15%, respectively. Samples were irradiated in a cobalt-60 gamma-irradiator (IR-221, Nordion International Ltd., Ontario, Canada) equipped with a 11.1 PBq source strength and operated at a dose rate of 10 kGy h 1. For maintaining temperature during irradiation, samples were placed in the container box filled with ice and dry ice for refrigeration and freezing conditions, respectively. Dosimetry was performed with 5-mm-diameter alanine dosimeters (Bruker Instruments, Rheinstetten, Germany). The actual doses at the center of the sample were within 72% of the target dose. The sample for the irradiation was put in a tube which was 15 cm in height and 5 cm in diameter. During the irradiation, the tube was being rotated for uniformity. 2.4. Titration of virus Following irradiation treatment, oysters were removed from pouches and the meat and liquor removed and homogenized by stomaching for 2 min. Approximately 1 ml of the homogenized samples was added to 9 ml MEM+antibiotics (final concentrations: penicillin, 1000 U/ml; streptomycin, 1000 ml/ml; amphotericin B, 2.5 ml/ml) and homogenized by stomaching for a further 2 min. These samples represented the 10 1 dilution used for further studies. Log dilutions (10 1–10 6) of samples were prepared in MEM containing 10% FBS, and 10 ml aliquots of each log dilution were added to 10 microtitre wells, containing cells. The plates were incubated at 37 1C in 5% CO2 for 5–6 days and examined daily for the development of viral CPE. Wells showing CPE were scored as virus-positive and the number of positive wells for each virus dilution was recorded and calculated as TCID50.
3. Results and discussion The effects of gamma irradiation on the levels of inactivation of poliovirus were compared in oyster and culture broth under different conditions (Table 1). Poliovirus could be readily inactivated by gamma irradiation in PBS buffer compared with oyster and culture broth. The D10 value of poliovirus in PBS buffer, virus culture medium and oyster was determined to 0.46, 2.84 and 2.94 kGy, respectively. The mechanism of virus inactivation by irradiation is mainly based on the reaction of OH free radicals with the nucleic acid strands, and the virus coat may also play a role. Gamma irradiation of viruses is less effective in the presence of organic materials which react with OH free radicals, the so-called scavengers (Sommer et al., 2001; Husman et al., 2004). This result shows the importance of the organic matters such as protein, sugar, and fat in virus inactivation by gamma irradiation. The initial PFU of poliovirus in culture broth was slightly decreased depending upon the decrease of pH and the increase of salt concentration, but the radiation sensitivity of poliovirus was not affected by pH and salt concentration (Table 1). Poliovirus is a member of the enterovirus subgroup, family Picornaviridae enteroviruses are transient inhabitants of the gastrointestinal tract, and are stable at acid pH. Ionic composition as well as pH has been reported to be important factors that influence the kinetics of viral inactivation in a variety of systems. In addition, the presence of organic matter in water directly or indirectly contributes to viral stability or inactivation (Alvarez et al., 2000). Eubanks and Farrah (1981) reported that poliovirus and other enteroviruses in sodium fluoride solution were rapidly inactivated, whereas other sodium salt had little or no effect on virus infectivity. In this study, sodium chloride at high concentration (15%) showed also a little effect on the inactivation of poliovirus. The present results suggest that the efficiency of gamma irradiation is inhibited by organic substances as they act as scavengers, pH and salt cannot affect to the effectiveness of gamma irradiation for inactivation of virus. The effects of irradiation temperature on the inactivation of poliovirus were also determined. The result showed that the initial PFU of poliovirus was not changed by refrigeration and freezing temperatures. However, the D10 value of poliovirus was dramatically increased at freezing temperature compared with room and refrigeration temperatures (Table 1). The radiation sensitivity of microorganisms is influenced by the composition of food substrate. The primary mode of action of ionizing radiation is the creation of hydroxyl and hydrogen radicals from water molecules. Under conditions of limited free water, such as in
ARTICLE IN PRESS P.-J. Jung et al. / Radiation Physics and Chemistry 78 (2009) 597–599
frozen state, higher radiation doses are typically required to reduce the microorganisms, with radiation resistance typically increasing to a maximum near 20 1C (Sommer et al., 2001; Thayer et al., 2001). In the present study, the radiation resistance of poliovirus was increased in the freezing temperature. From these results, it was revealed that the presence of organic substance and irradiation temperature were the important factors for the inactivation of viruses by gamma irradiation, but pH and salt had a little effect on the effectiveness of gamma irradiation.
Acknowledgements This research was supported by Nuclear Research & Development Program of the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean government (MEST). References Alvarez, M.E., Aguilar, M., Fountain, A., Gonzalez, N., Rascon, O., Saenz, D., 2000. Inactivation of MS-2 phage and poliovirus in groundwater. Journal of Microbiology 46, 159–165.
599
Duizer, E., Schwab, K.J., Neill, F.H., Atmar, R.L., Koopmans, M.P.G., Estes, M.K., 2004. Laboratory efforts to cultivate noroviruses. Journal of General Virology 85, 79–87. Gillespie, J.A., Adak, G.K., O’Brien, S.J., Brett, M.M., Bolton, F.J., 2001. General outbreaks of infectious disease associated with fish and shellfish, England and Wales, 1992–1999. Communicable Disease and Public Health 4, 117–123. Husman, A.M.R., Bijkerk, P., Lodder, W., Berg, H., Pribil, W., Cabaj, A., Gehriger, P., Sommer, R., Duizer, E., 2004. Calicivirus inactivation by nonionizing (253.7-nanometer-wavelength [UV] and ionizing (Gamma) radiation. Applied Microbiology 70, 5089–5093. Kester, D.R., Pytkowicz, R.M., 1967. Determination of the apparent dissociation constants of acid in seawater. Limnology and Oceanography 12, 243–252. Lees, D., 2000. Viruses and bivalve shellfish. International Journal of Food Microbiology 59, 81–116. Potasman, I., Paz, A., Odeh, M., 2002. Infectious outbreaks associated with bivalve shellfish consumption: a worldwide perspective. Clinical Infectious Diseases 35, 921–928. Slomka, M.J., Appleton, H., 1998. Feline calicivirus as a model system for heat inactivation studies of small round structured viruses in shellfish. Epidemiology and Infection 121, 401–407. Sommer, R., Pribil, W., Appelt, S., Gehringer, P., Eschweiler, H., Leth, H., Cabaj, A., Haider, T., 2001. Inactivation of bacteriophages in water by means of nonionizing (UV-253.7 nm) and ionizing (gamma) radiation: a comparative approach. Water Research 35, 3109–3116. Sumner, J., Ross, T., 2002. A semi-quantitative seafood safety risk assessment. International Journal of Food Microbiology 77, 55–59. Thayer, D.W., Boyd, G., 2001. Effect of irradiation temperature on inactivation of Escherichia coli O157: H7 and Staphylococcus aureus. Journal of Food Protection 64, 1624–1626.
Contents lists available at ScienceDirect
Radiation Physics and Chemistry journal homepage: www.elsevier.com/locate/radphyschem
Radiation sensitivity of poliovirus, a model for norovirus, inoculated in oyster (Crassostrea gigas) and culture broth under different conditions Pil-Mun Jung a, Jae Seok Park b, Jin-Gyu Park a, Jae-Nam Park a, In-Jun Han a, Beom-Seok Song a, Jong-il Choi a, Jae-Hun Kim a, Myung-Woo Byun a, Min Baek c, Young-Jin Chung d, Ju-Woon Lee a, a
Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185, South Korea Korea Food & Drug Administration, Seoul 122-704, South Korea c Atomic Energy Policy Division, Ministry of Education, Science and Technology, Gwacheon 427-715, South Korea d Department of Food and Nutrition, Chungnam National University, Daejeon 305-764, South Korea b
a r t i c l e in fo
Keywords: Radiation sensitivity Poliovirus Oyster Gamma irradiation
abstract Poliovirus is a recognized surrogate for norovirus, pathogen in water and food, due to the structural and genetic similarity. Although radiation sensitivity of poliovirus in water or media had been reported, there has been no research in food model such as shellfish. In this study, oyster (Crassostrea gigas) was incubated in artificial seawater contaminated with poliovirus, and thus radiation sensitivity of poliovirus was determined in inoculated oyster. The effects of ionizing radiation on the sensitivity of poliovirus were also evaluated under different conditions such as pH (4–7) and salt concentration (1–15%) in culture broth, and temperature during irradiation. The D10 value of poliovirus in PBS buffer, virus culture broth and oyster was determined to 0.46, 2.84 and 2.94 kGy, respectively. The initial plaque forming unit (PFU) of poliovirus in culture broth was slightly decreased as the decrease of pH and the increase of salt concentration, but radiation sensitivity was not affected by pH and salt contents. However, radiation resistance of poliovirus was increased at frozen state. These results provide the basic information for the inactivation of pathogenic virus in foods by using irradiation. & 2009 Published by Elsevier Ltd.
1. Introduction Despite the introduction of sanitary controls regulating their production, shellfish, particularly bivalve molluscs, continue to feature highly in statistics of food-borne disease (Potasman et al., 2002; Sumner and Ross, 2002). Recent decades have also seen a significant change in the reported etiology of shellfish-borne illness. Epidemiological evidence suggests that human enteric viruses, notably norovirus, formerly ‘norwalk-like virus’ or small round structured viruses, and hepatitis A virus (HAV), are now the most common cause of shellfish-borne illness (Gillespie et al., 2001; Lees, 2000). These changes may be explained, in part, by the increased surveillance for these viruses and recognition of their importance in terms of human health, but it is also likely to reflect limitations in the current production and purification processes used for shellfish. Further processing may, therefore, be required to improve the safety of shellfish for human consumption. However, shellfish such as oysters and clams are often eaten raw or lightly cooked and this not only has implications for disease transmission, but also limits processing methods suitable
for these products. The preservation methods, such as highpressure and thermal processing, can be used to eliminate a pathogenic virus (Slomka and Appleton, 1998). However, these processes will not only alter the sensory and nutritional qualities of the food, but convert the product into cooked oysters. Irradiation treatment is being used more frequently as a useful and effective means of disinfection without compromising sensory and nutritional quality (Sommer et al., 2001). Poliovirus is recognized as a surrogate for norovirus used in inactivation studies, because norovirus cannot be cultured (Duizer et al., 2004). Although radiation sensitivity of poliovirus in water or media had been reported (Husman et al., 2004), there was no research in food model such as shellfish. Therefore, the aim of this study was to determine the radiation sensitivity of poliovirus inoculated in oyster and evaluate the effect of ionizing radiation on the sensitivity of poliovirus under different conditions.
2. Materials and methods 2.1. Cell and virus culture
Corresponding author. Tel.: +82 63 570 3204; fax: +82 63 570 3207.
E-mail address: [email protected] (J.-W. Lee). 0969-806X/$ - see front matter & 2009 Published by Elsevier Ltd. doi:10.1016/j.radphyschem.2009.03.017
LLC-MK2 (ATCC number: CCL-7.1TM) cell was grown in 75 cm2 plastic cell culture flasks in Eagle’s minimal essential medium
ARTICLE IN PRESS 598
P.-J. Jung et al. / Radiation Physics and Chemistry 78 (2009) 597–599
(MEM) supplemented with 10% irradiated fetal bovine serum (FBS). Poliovirus, serotype poliovirus-1 (ATCC VR-1562TM) was propagated in LLC-MK2 monolayer. Flask monolayer cultures were inoculated with LLC-MK2 cell incubated until a visible cytopathic effect (CPE) affecting approximately 80% of the cells was observed. The cultures were then frozen and thawed three times, centrifuged at 1000g for 20 min to remove cell debris, and the supernatants pooled and stored at 70 1C prior to use.
Table 1 Radiosensitivity (D10) of poliovirus in various irradiation conditions. Irradiation condition
D10 value (kGy)
Oyster Culture broth PBS buffer pH
7.0 6.0 5.0 4.0
2.84 2.94 0.46 3.71 3.40 3.73 3.39
Salt concentration (%)
1.0 3.5 7.0 15.0
2.94 3.01 3.11 3.14
Irradiation temperature (1C)
Room temp. Refrigeration on ice Freezing on dry ice
3.39 3.22 8.25
2.2. Shellfish maintenance and inoculation of poliovirus Artificial seawater was prepared according to the method described by Kester and Pytkowicz (1967) and this artificial seawater was then added to commercial depuration tank. The depuration system was run overnight to aerate the water and stabilize the temperature at 17 1C. Oysters (Crassostrea gigas) were obtained from Tongyeong Oyster Fishery, Tongyeong City, Gyeongsangnam-do, Korea. The oysters were washed to remove debris, placed in plastic breadbaskets, and transferred to the depuration tank for 48 h prior to use. The natural accumulation of viruses by shellfish was simulated by incubating batches of oysters at 16 1C in the dark for 2 h in 7 L artificial seawater to which was added 50 ml poliovirus stock with an infectivity titer of 8.0870.15 TCID50 (tissue culture infectious dose, log10). This resulted in seawater containing 4.2370.33 TCID50 of poliovirus. Air was pumped via an aquarium pump into the seawater to ensure adequate aeration throughout the inoculation period. 2.3. Sample preparation and gamma irradiation To adjust pH of salt concentration in culture broth, 5 M HCl and 1 M NaOH and NaCl were used to make virus suspension in MEM covering the pH range from 4 to 7 and the salt concentration range from 1% to 15%, respectively. Samples were irradiated in a cobalt-60 gamma-irradiator (IR-221, Nordion International Ltd., Ontario, Canada) equipped with a 11.1 PBq source strength and operated at a dose rate of 10 kGy h 1. For maintaining temperature during irradiation, samples were placed in the container box filled with ice and dry ice for refrigeration and freezing conditions, respectively. Dosimetry was performed with 5-mm-diameter alanine dosimeters (Bruker Instruments, Rheinstetten, Germany). The actual doses at the center of the sample were within 72% of the target dose. The sample for the irradiation was put in a tube which was 15 cm in height and 5 cm in diameter. During the irradiation, the tube was being rotated for uniformity. 2.4. Titration of virus Following irradiation treatment, oysters were removed from pouches and the meat and liquor removed and homogenized by stomaching for 2 min. Approximately 1 ml of the homogenized samples was added to 9 ml MEM+antibiotics (final concentrations: penicillin, 1000 U/ml; streptomycin, 1000 ml/ml; amphotericin B, 2.5 ml/ml) and homogenized by stomaching for a further 2 min. These samples represented the 10 1 dilution used for further studies. Log dilutions (10 1–10 6) of samples were prepared in MEM containing 10% FBS, and 10 ml aliquots of each log dilution were added to 10 microtitre wells, containing cells. The plates were incubated at 37 1C in 5% CO2 for 5–6 days and examined daily for the development of viral CPE. Wells showing CPE were scored as virus-positive and the number of positive wells for each virus dilution was recorded and calculated as TCID50.
3. Results and discussion The effects of gamma irradiation on the levels of inactivation of poliovirus were compared in oyster and culture broth under different conditions (Table 1). Poliovirus could be readily inactivated by gamma irradiation in PBS buffer compared with oyster and culture broth. The D10 value of poliovirus in PBS buffer, virus culture medium and oyster was determined to 0.46, 2.84 and 2.94 kGy, respectively. The mechanism of virus inactivation by irradiation is mainly based on the reaction of OH free radicals with the nucleic acid strands, and the virus coat may also play a role. Gamma irradiation of viruses is less effective in the presence of organic materials which react with OH free radicals, the so-called scavengers (Sommer et al., 2001; Husman et al., 2004). This result shows the importance of the organic matters such as protein, sugar, and fat in virus inactivation by gamma irradiation. The initial PFU of poliovirus in culture broth was slightly decreased depending upon the decrease of pH and the increase of salt concentration, but the radiation sensitivity of poliovirus was not affected by pH and salt concentration (Table 1). Poliovirus is a member of the enterovirus subgroup, family Picornaviridae enteroviruses are transient inhabitants of the gastrointestinal tract, and are stable at acid pH. Ionic composition as well as pH has been reported to be important factors that influence the kinetics of viral inactivation in a variety of systems. In addition, the presence of organic matter in water directly or indirectly contributes to viral stability or inactivation (Alvarez et al., 2000). Eubanks and Farrah (1981) reported that poliovirus and other enteroviruses in sodium fluoride solution were rapidly inactivated, whereas other sodium salt had little or no effect on virus infectivity. In this study, sodium chloride at high concentration (15%) showed also a little effect on the inactivation of poliovirus. The present results suggest that the efficiency of gamma irradiation is inhibited by organic substances as they act as scavengers, pH and salt cannot affect to the effectiveness of gamma irradiation for inactivation of virus. The effects of irradiation temperature on the inactivation of poliovirus were also determined. The result showed that the initial PFU of poliovirus was not changed by refrigeration and freezing temperatures. However, the D10 value of poliovirus was dramatically increased at freezing temperature compared with room and refrigeration temperatures (Table 1). The radiation sensitivity of microorganisms is influenced by the composition of food substrate. The primary mode of action of ionizing radiation is the creation of hydroxyl and hydrogen radicals from water molecules. Under conditions of limited free water, such as in
ARTICLE IN PRESS P.-J. Jung et al. / Radiation Physics and Chemistry 78 (2009) 597–599
frozen state, higher radiation doses are typically required to reduce the microorganisms, with radiation resistance typically increasing to a maximum near 20 1C (Sommer et al., 2001; Thayer et al., 2001). In the present study, the radiation resistance of poliovirus was increased in the freezing temperature. From these results, it was revealed that the presence of organic substance and irradiation temperature were the important factors for the inactivation of viruses by gamma irradiation, but pH and salt had a little effect on the effectiveness of gamma irradiation.
Acknowledgements This research was supported by Nuclear Research & Development Program of the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean government (MEST). References Alvarez, M.E., Aguilar, M., Fountain, A., Gonzalez, N., Rascon, O., Saenz, D., 2000. Inactivation of MS-2 phage and poliovirus in groundwater. Journal of Microbiology 46, 159–165.
599
Duizer, E., Schwab, K.J., Neill, F.H., Atmar, R.L., Koopmans, M.P.G., Estes, M.K., 2004. Laboratory efforts to cultivate noroviruses. Journal of General Virology 85, 79–87. Gillespie, J.A., Adak, G.K., O’Brien, S.J., Brett, M.M., Bolton, F.J., 2001. General outbreaks of infectious disease associated with fish and shellfish, England and Wales, 1992–1999. Communicable Disease and Public Health 4, 117–123. Husman, A.M.R., Bijkerk, P., Lodder, W., Berg, H., Pribil, W., Cabaj, A., Gehriger, P., Sommer, R., Duizer, E., 2004. Calicivirus inactivation by nonionizing (253.7-nanometer-wavelength [UV] and ionizing (Gamma) radiation. Applied Microbiology 70, 5089–5093. Kester, D.R., Pytkowicz, R.M., 1967. Determination of the apparent dissociation constants of acid in seawater. Limnology and Oceanography 12, 243–252. Lees, D., 2000. Viruses and bivalve shellfish. International Journal of Food Microbiology 59, 81–116. Potasman, I., Paz, A., Odeh, M., 2002. Infectious outbreaks associated with bivalve shellfish consumption: a worldwide perspective. Clinical Infectious Diseases 35, 921–928. Slomka, M.J., Appleton, H., 1998. Feline calicivirus as a model system for heat inactivation studies of small round structured viruses in shellfish. Epidemiology and Infection 121, 401–407. Sommer, R., Pribil, W., Appelt, S., Gehringer, P., Eschweiler, H., Leth, H., Cabaj, A., Haider, T., 2001. Inactivation of bacteriophages in water by means of nonionizing (UV-253.7 nm) and ionizing (gamma) radiation: a comparative approach. Water Research 35, 3109–3116. Sumner, J., Ross, T., 2002. A semi-quantitative seafood safety risk assessment. International Journal of Food Microbiology 77, 55–59. Thayer, D.W., Boyd, G., 2001. Effect of irradiation temperature on inactivation of Escherichia coli O157: H7 and Staphylococcus aureus. Journal of Food Protection 64, 1624–1626.