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One-year monthly monitoring of Torque teno virus (TTV) in wastewater treatment plants in Japan
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Water Research 39 (2005) 2008–2013 www.elsevier.com/locate/watres
One-year monthly monitoring of Torque teno virus (TTV) in wastewater treatment plants in Japan Eiji Haramotoa,, Hiroyuki Katayamaa, Kumiko Ogumaa, Hiromasa Yamashitab, Eiichiro Nakajimab, Shinichiro Ohgakia a
Department of Urban Engineering, School of Engineering, University of Tokyo, Bunkyo-ku, Tokyo 113–8656, Japan b Water Quality Control Department, National Institute for Land and Infrastructure Management, Tsukuba-shi, Ibaraki 305–0804, Japan Received 26 October 2004; received in revised form 21 January 2005; accepted 3 March 2005
Abstract Torque teno virus (TTV) is a novel hepatitis virus which is considered to be transmitted by the fecal-oral route. Wastewater samples were collected monthly from eight wastewater treatment plants in Japan for 1 year, from July 2003 to June 2004, and tested for the presence of TTV by TaqMan PCR. TTV was detected in 97% (93/96) of influent samples, implying that TTV is epidemic in Japan. TTV was also isolated in 18% (17/96) of secondary effluent samples before chlorination and in 24% (23/95) of final effluent samples after chlorination. There was no significant difference between the concentration of total coliform in TTV-positive final effluents and that in TTV-negative final effluents, which indicates that total coliform cannot be used as an indicator of TTV. No TTV was detected in 24 effluents for reuse from two wastewater treatment plants using sand filtration and ozonation. r 2005 Elsevier Ltd. All rights reserved. Keywords: Wastewater treatment plant; Torque teno virus; Total coliform; Virus concentration; TaqMan PCR
1. Introduction Torque teno virus (TTV) was first isolated in 1997 in a Japanese patient with posttransfusion hepatitis of unknown etiology (Nishizawa et al., 1997). It has a diameter of 30–32 nm (Itoh et al., 2000) and posses a non-enveloped, single-stranded and circular DNA (Mushahwar et al., 1999; Okamoto et al., 1998a). TTV is tentatively classified as the genus Anellovirus (Okamoto et al., 2004), and divided into five phylogenetic Corresponding author. Tel.: +81 3 5841 6242;
fax: +81 3 5841 6244. E-mail address: [email protected] (E. Haramoto).
groups consisting of more than 30 genotypes (Heller et al., 2001; Muljono et al., 2001; Okamoto et al., 1999, 2001; Peng et al., 2002). TTV is prevalent worldwide in general populations, such as blood donors, as well as in patients of hepatitis (Okamoto et al., 1998a; Biagini, 2001; Iriyama, 1999; Kato, 2000; Niel, 1999; Takahashi et al., 1998). TTV was originally suspected to cause hepatitis, but recent studies have indicated that it may not cause serious health problems (Abe et al., 1999). However, several genotypes of TTV are thought to be responsible for human diseases (Takahashi et al., 1998; Umemura et al., 2001). TTV is excreted in feces as a complete virion, suggesting that the fecal-oral route is a predominant mode of its transmission (Okamoto et al., 1998b).
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The TaqMan PCR quantification system has been developed and applied to the detection of TTV by many researchers (Iriyama et al., 1999; Kato et al., 2000; Takahashi et al., 1998; Moen et al., 2002; Tokita et al., 2002). Primers based on the sequence of a coding region can be used to detect TTV of group 1, while TTV of all known groups can be detected using primers based on the sequence of a non-coding region (Okamoto et al., 1998b, 2000). In order to clarify the infection route of TTV, it is important to accumulate the information on the occurrence of TTV in the environment. TTV has been successfully detected in 13% (8/63) and 2% (1/48) of 40 ml influent and effluent samples from a wastewater treatment plant (WWTP) in India, respectively (Vaidya et al., 2002). It has been also isolated from 8% (9/113) of shellfish samples in Norway (Myrmel et al., 2004). In our previous study, 3 (5%) of 64 river water samples in Japan showed positive for TTV (Haramoto et al., 2005). In this study, four kinds of wastewater samples (influent, secondary effluent, final effluent, and effluent for reuse) were collected monthly from eight WWTPs in Japan for 1 year, from July 2003 to June 2004, and applied to the detection of TTV by using TaqMan PCR. The concentration of total coliform (TC), which is currently used as an indicator microorganism in the effluents from WWTPs in Japan, was also tested, and the relationship between TC and TTV was determined.
2. Materials and methods 2.1. Collection of wastewater samples Eight WWTPs (named WWTPs A to H) were selected from seven prefectures in Japan: two WWTPs in Tokyo prefecture (WWTPs A and B), and one WWTP each in Kanagawa (WWTP C), Saitama (WWTP D), Ibaraki (WWTP E), Osaka (WWTP F), Kyoto (WWTP G), and Shiga (WWTP H) prefectures. These WWTPs treat 1.0 107 to 2.1 108 m3 of sewage per year with an average of 5.8 107 m3 per year. A standard activated sludge process is installed at WWTPs A, B, D, F, and G. Meanwhile, an anaerobic-oxic (AO) or an anaerobicanoxic-oxic (A2O) activated sludge process is installed at WWTP C or H, respectively. WWTP E is equipped with a nitrifying-denitrifying activated sludge process. At each WWTP, influent samples, secondary effluent samples before chlorination, and final effluent samples after chlorination were collected once a month for 1 year, from July 2003 to June 2004. In order to obtain sufficient contact time with chlorine, the final effluent samples were kept at room temperature for 15 min after the sampling, followed by the addition of sodium thiosulfates for the neutralization of chlorine. In total,
2009
96 influent, 96 secondary effluent, and 95 final effluent samples were analyzed. Due to the failure of the sampling, the final effluent sample from WWTP H was not collected in October 2003. At WWTPs B and C, effluent samples for reuse, treated with sand filtration and ozonation, were also collected in addition to the other samples. All samples were collected and stored in plastic bottles on ice. The samples were delivered to the laboratory within 1 day after collection and analyzed immediately upon arrival. All the samples were assayed for TC by a single agar layer method using chromocult coliform agar (Merck, Tokyo, Japan) according to the protocols described by the manufacture. Suspended solid (SS) was also measured following the standard methods of the examination of water in Japan (Japan Water Works Associtation, 1993). Temperature and pH of the samples were determined on site upon collection. 2.2. Concentration of wastewater samples The method used for the concentration of wastewater samples was described previously (Katayama et al., 2002). Briefly, 2.5 M MgCl2 was inoculated into the sample to obtain a concentration of 25 mM, then the sample was passed through an HA filter (0.45 mm pore size and 90 mm diameter; Millipore, Tokyo, Japan) attached to a glass filter holder (Advantec, Tokyo, Japan). The volumes filtered were 100 ml for influent, and 1000 ml for secondary effluent, final effluent, and effluent for reuse. The filter was rinsed with 200 ml of 0.5 mM H2SO4 (pH 3.0), followed by elution of viruses with 10 ml of 1 mM NaOH (pH 10.8). The filtrate was recovered in a tube containing 50 ml of 100 mM H2SO4 (pH 1.0) and 100 ml of 100 TrisEDTA buffer (pH 8.0) for neutralization, followed by ultrafiltration using a Centriprep YM-50 (Millipore) at 2500 rpm for 10 min. The sample which did not pass through an ultrafiltration membrane of the Centriprep YM-50 (approximately 2 ml) was applied to further centrifugation at 2500 rpm for 5 min to obtain a final volume of 700 ml. 2.3. DNA extraction and TaqMan PCR Viral DNA was extracted from 200 ml of the final concentrated sample using a QIAamp DNA mini kit (Qiagen, Tokyo, Japan) according to the protocols described by the manufacture. Five microliters out of 200 ml of the resulting extracted DNA was mixed with 45 ml of a reaction buffer containing 25 ml of 2 TaqMan universal PCR master mix (Applied Biosystems, Tokyo, Japan), 2 ml of 10 mM sense primer (50 -CGGGTGCCGDAGGTGAGTTTACAC-30 ), 2 ml of 10 mM antisense primer (50 -GAGCCTTGCCCATRGCCCGGCCAG-30 ), and 3 ml of 5 mM TaqMan probe
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2010
(50 -FAM [6-carboxyfluorescein]-AGTCAAGGGGCAATTCGGGCTCGGGA-TAMRA [6-carboxytetramethylrhodamine]-30 ) (Tokita et al., 2002), where R is A or G; and D is A, G, or T. Subsequently, the mixture was added to a well of a 96 well micro plate (Applied Biosystems). The plate was incubated at 50 1C for 2 min and 95 1C for 10 min, followed by 50 cycles at 95 1C for 10 s and at 62 1C for 30 s, and it was finally cooled to 4 1C. The DNA amplification was determined using the ABI PRISM 7000 sequence detection system (Applied Biosystems). Three wells were adopted for the detection of TTV for one sample. A positive result was obtained when at least one of the three wells showed the DNA amplification. Two hundred microliters out of 700 ml of the final concentrated sample was applied to DNA extraction, and 15 ml out of 200 ml of the extracted DNA was used for the detection of TTV. Therefore, volumes tested for the presence of TTV were equivalent to approximately 2.1 ml for influent, and 21 ml for secondary effluent, final effluent, and effluent for reuse.
3. Results 3.1. Influent samples Table 1 shows the water quality parameters of wastewater samples. Temperatures of the samples collected in winter were usually lower than 20 1C, while those collected in summer sometimes exceeded 25 1C. The concentration of TC in the influent ranged from 80,000 to 4,100,000 CFU/ml, but the monthly mean values of TC did not show any seasonal variation. As summarized in Table 2, TTV was detected in 93 of 96 influent samples, positive for 97%. 3.2. Secondary and final effluent samples Secondary and final effluent samples were collected before and after chlorination, respectively. The annual mean values of TC were 680 CFU/ml (range: 0–5400 CFU/ml) and 130 CFU/ml (range: 6.5–2800 CFU/ml) for the secondary and final effluent
Table 1 Water quality of wastewater samples Sample type (no. of samples)a
Influent (96) Secondary effluent (96) Final effluent (95) Effluent for reuse (24)
Mean (range) Temperature (1C)
pH
21 22 22 22
7.4 6.7 6.7 7.1
(14–28) (15–28) (15–29) (17–27)
(6.6–8.4) (6.3–7.7) (6.3–7.4) (6.7–7.5)
SS (mg/l)
TC (CFU/ml)
190 (67–560) 1.9 (0–6.5) 1.9 (0–12) 2.8 (0–23)
360,000 (80,000–4,100,000) 680 (0–5,400) 130 (6.5–2,800) 6.0 (0–54)
a Influent, secondary effluent, and final effluent samples were collected from eight WWTPs, and effluent for reuse were collected from two of eight WWTPs.
Table 2 Detection of TTV in wastewater samples Time of collection (year and month)
2003
2004
Total (% positive)
July Aug. Sep. Oct. Nov. Dec. Jan. Feb. Mar. Apr. May June
No. of positive samples/no. of samples tested Influent
Secondary effluent
Final effluent
Effluent for reuse
8/8 8/8 8/8 7/8 8/8 7/8 8/8 7/8 8/8 8/8 8/8 8/8
2/8 2/8 0/8 2/8 2/8 2/8 0/8 0/8 1/8 2/8 3/8 1/8
3/8 3/8 3/8 2/7 2/8 0/8 2/8 3/8 1/8 1/8 2/8 1/8
0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2
93/96 (97%)
17/96 (18%)
23/95 (24%)
0/24 (0%)
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3.3. Effluent samples for reuse Effluents for reuse were collected from two (WWTPs B and C) out of eight WWTPs. The advanced wastewater treatment process using sand filtration followed by ozonation did not remove SS effectively but reduced TC more than 1.4 log (Table 1). All 24 effluents for reuse were negative for TTV, while it was detected in 6 (25%) of 24 final effluents collected from WWTPs B and C (Table 2). 3.4. Difference among WWTPs The difference of the TC concentrations in influent and final effluent samples among eight WWTPs are shown in Fig. 1. The annual mean concentration of TC in the influent was similar for all WWTPs, ranging from 210,000 (WWTP G) to 560,000 CFU/ml (WWTP B). The annual mean concentration of TC in the final effluent collected from each WWTP ranged from 23 (WWTP H) to 460 CFU/ml (WWTP C). Out of 12 final effluent samples collected from each WWTP, TTV was detected twice (17%), three times (25%), and four times (33%) at two (WWTPs A and B), one (WWTP E), and four (WWTPs C, D, F, and G) WWTPs, respectively. However, all 11 final effluent samples collected from WWTP H were negative for TTV.
4. Discussion TTV is an emerging virus which was discovered as an agent of unknown hepatitis in Japan in 1997. TTV is isolated not only in the patients of hepatitis but also in the healthy persons (Okamoto et al., 1998a; Biagini et al., 2001; Iriyama et al., 1999; Kato et al., 2000; Niel et al., 1999; Takahashi et al., 1998), thus it is strongly
Influent 7
Final effluent
6 TC (log CFU/mL)
samples, respectively (Table 1). The annual mean inactivation ratio of TC by chlorination was 0.71 log with a maximum value of 2.2 log. On the other hand, chlorination did not affect the results of PCR for TTV. TTV was detected in 18% (17/96) and 24% (23/95) of secondary and final effluent samples, respectively (Table 2). Comparison between the influent and effluent samples revealed that 89–100% of SS were removed by the wastewater treatment. The total reduction ratio of TC throughout the treatment ranged from 2.0 to 5.0 log with an average of 3.4 log. The mean concentration of TC was 230 CFU/ml (range: 20–2800 CFU/ml) for TTVpositive final effluent samples, while it was 140 CFU/ml (range: 6.5–1400 CFU/ml) for TTV-negative final effluent samples.
2011
5 4 3 2 1 0
A
B
C
D
E
F
G
H
WWTP Fig. 1. Difference of TC concentrations among eight WWTPs. The plot, upper bar, and lower bar show the geometric mean, maximum, and minimum data of TC concentrations in 11 or 12 the samples collected for 1 year, respectively.
suspected to be transmitted by the fecal-oral route (Okamoto et al., 1998b). There are only a few studies on the detection of TTV in the environmental samples: influent and effluent samples from a WWTP in India (Vaidya et al., 2002), shellfish samples in Norway (Myrmel et al., 2004), and river water samples in Japan (Haramoto et al., 2005). In this study, the occurrence of TTV in wastewater samples collected from eight WWTPs in Japan was surveyed for 1 year. The samples were concentrated using the method developed in our laboratory (Katayama et al., 2002), followed by TaqMan PCR using the primer pairs and the TaqMan probe specific for TTV of all known genotypes (Tokita et at., 2002). In total, TTV was detected in 97% (93/96), 18% (17/96), or 24% (23/ 95) of influent, secondary effluent, or final effluent samples, respectively, while no TTV was detected in 24 effluent samples for reuse (Table 2). The presence or absence of TTV was tested by using only small volumes which were equivalent to approximately 2.1 ml (influent) or 21 ml (secondary effluent, final effluent, and effluent for reuse) of the original samples. Compared with the study conducted by Vaidya et al. in India (Vaidya et al., 2002), this study showed a very high positive ratio of TTV from influent samples. This might be explained by the fact that TTV was isolated in nearly 90% of blood donors in Japan (Kato et al., 2000; Takahashi et al., 1998). Moreover, the high positive ratio of TTV observed in this study might also be attributable to our detection method which possibly has an advantage at the detection of TTV including the high recovery efficiency in the concentration process, and/or the high sensitivity of the TaqMan PCR system. TC was also tested in this study because it is an indicator microorganism in the standard for the
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discharge from a WWTP in Japan: the concentration of TC must be lower than 3000 CFU/ml. The concentrations of TC in wastewater samples were stable throughout the year. That is, the mean values of the concentrations of TC were 360,000, 680, 130, and 6.0 CFU/ml for influent, secondary effluent, final effluent, and effluent for reuse, respectively (Table 1). All 95 final effluent samples met the standard for TC. Chlorination achieved a 0.71 log reduction of TC on the average, but the positive ratio of TTV was not decreased by chlorination (Table 2). There is no data on the tolerance of TTV to chlorine, but it is known that the genomes of enteric viruses, such as poliovirus and hepatitis A virus, inactivated by chlorine can be detected by PCR without any specific treatment (Nuanualsuwan and Cliver, 2002). Therefore, the PCR test adopted in this study may have detected the DNA of chlorine-inactivated TTV. Further studies are required to evaluate the resistance of TTV to chlorine. There was no significant difference between the concentration of TC in TTV-positive final effluents and that in TTV-negative final effluents (t-test, P40.05), implying a possibility that TC may not indicate the fate of TTV discharged from WWTPs to the water environment. The annual mean reduction ratio of TC at WWTP H was 4.3 log, while those at other seven WWTPs ranged from 2.8 to 3.8 log (Fig. 1). TTV was not detected in effluent samples collected from WWTP H but detected in 17–33% of effluent samples collected from other seven WWTPs. These results suggest that the A2O activated sludge process conducted at WWTP H is an effective system to reduce the concentration of bacteria and viruses, including TC and TTV, in the influent. The advanced wastewater treatment using sand filtration and ozonation was also considered as a proper method to remove them. Monitoring of viruses in the influent of a WWTP provides useful information to determine their actual incidence in its service area. From the results in this study, it is suggested that TTV is epidemic in Japan throughout the year and that TTV is continuously discharged into the water environment after the treatment at WWTPs. The fate of TTV discharged from WWTPs need to be studied in the future.
5. Conclusion TTV, an emerging hepatitis virus, was detected in 97% (93/96), 18% (17/96), 24% (23/95), and 0% (0/24) of influents, secondary effluents, final effluents, and effluents for reuse collected from eight WWTPs in Japan, respectively. No clear correlation was observed between TTV and TC in the final effluents, implying the need for further studies on the appropriate indicators of
TTV. The high incidence of TTV in the influents indicated that the virus is epidemic widely in Japan throughout the year.
Acknowledgements This study was supported by the Microbial Water Quality Project on Treated Sewage and Reclaimed Wastewater, Ministry of Land, Infrastructure and Transport Government of Japan. We also thank Mr. Atsushi Tajima and Mr. Kensuke Sakurai (National Institute for Land and Infrastructure Management, Ibaraki, Japan), and Ms. Hozue Kuroda (University of Tokyo, Tokyo, Japan), for their help.
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ARTICLE IN PRESS E. Haramoto et al. / Water Research 39 (2005) 2008–2013 2000. Development of a TT virus DNA quantification system using real-time detection PCR. J. Clin. Microbiol. 38, 94–98. Moen, E.M., Sleboda, J., Grinde, B., 2002. Real-time PCR methods for independent quantitation of TTV and TLMV. J. Virol. Methods. 104, 59–67. Muljono, D.H., Nishizawa, T., Tsuda, F., Takahashi, M., Okamoto, H., 2001. Molecular epidemiology of TT virus (TTV) and characterization of two novel TTV genotypes in Indonesia. Arch. Virol. 146, 1249–1266. Mushahwar, I.K., Erker, J.C., Muerhoff, A.S., Leary, T.P., Simons, J.N., Birkenmeyer, L.G., Chalmers, M.L., PilotMatias, T.J., Dexai, S.M., 1999. Molecular and biophysical characterization of TT virus: evidence for a new virus family infecting humans. Proc. Natl. Acad. Sci. USA 96, 3177–3182. Myrmel, M., Berg, E.M.M., Rimstad, E., Grinde, B., 2004. Detection of enteric viruses in shellfish from the Norwegian coast. Appl. Environ. Microbiol. 70, 2678–2684. Niel, C., de Oliveira, J.M., Ross, R.S., Gomes, S.A., Roggendorf, M., Viazov, S., 1999. High prevalence of TT virus infection in Brazilian blood donors. J. Med. Virol. 57, 259–263. Nishizawa, T., Okamoto, H., Konishi, K., Yoshizawa, H., Miyakawa, Y., Mayumi, M., 1997. A novel DNA virus (TTV) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology. Biochem. Biophys. Res. Commun. 241, 92–97. Nuanualsuwan, S., Cliver, D.O., 2002. Pretreatment to avoid positive RT-PCR results with inactivated viruses. J. Virol. Methods 104, 217–225. Okamoto, H., Nishizawa, T., Kato, N., Ukita, M., Ikeda, H., Iizuka, H., Miyakawa, Y., Mayumi, M., 1998a. Molecular cloning and characterization of a novel DNA virus (TTV) associated with posttransfusion hepatites of unknown etiology. Hepatol. Res. 10, 1–16. Okamoto, H., Akahane, Y., Ukita, M., Fukuda, M., Tsuda, F., Miyakawa, Y., Mayumi, M., 1998b. Fecal excretion of a nonenveloped DNA virus (TTV) associated with posttransfusion non-A-G hepatitis. J. Med. Virol. 56, 128–132.
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One-year monthly monitoring of Torque teno virus (TTV) in wastewater treatment plants in Japan Eiji Haramotoa,, Hiroyuki Katayamaa, Kumiko Ogumaa, Hiromasa Yamashitab, Eiichiro Nakajimab, Shinichiro Ohgakia a
Department of Urban Engineering, School of Engineering, University of Tokyo, Bunkyo-ku, Tokyo 113–8656, Japan b Water Quality Control Department, National Institute for Land and Infrastructure Management, Tsukuba-shi, Ibaraki 305–0804, Japan Received 26 October 2004; received in revised form 21 January 2005; accepted 3 March 2005
Abstract Torque teno virus (TTV) is a novel hepatitis virus which is considered to be transmitted by the fecal-oral route. Wastewater samples were collected monthly from eight wastewater treatment plants in Japan for 1 year, from July 2003 to June 2004, and tested for the presence of TTV by TaqMan PCR. TTV was detected in 97% (93/96) of influent samples, implying that TTV is epidemic in Japan. TTV was also isolated in 18% (17/96) of secondary effluent samples before chlorination and in 24% (23/95) of final effluent samples after chlorination. There was no significant difference between the concentration of total coliform in TTV-positive final effluents and that in TTV-negative final effluents, which indicates that total coliform cannot be used as an indicator of TTV. No TTV was detected in 24 effluents for reuse from two wastewater treatment plants using sand filtration and ozonation. r 2005 Elsevier Ltd. All rights reserved. Keywords: Wastewater treatment plant; Torque teno virus; Total coliform; Virus concentration; TaqMan PCR
1. Introduction Torque teno virus (TTV) was first isolated in 1997 in a Japanese patient with posttransfusion hepatitis of unknown etiology (Nishizawa et al., 1997). It has a diameter of 30–32 nm (Itoh et al., 2000) and posses a non-enveloped, single-stranded and circular DNA (Mushahwar et al., 1999; Okamoto et al., 1998a). TTV is tentatively classified as the genus Anellovirus (Okamoto et al., 2004), and divided into five phylogenetic Corresponding author. Tel.: +81 3 5841 6242;
fax: +81 3 5841 6244. E-mail address: [email protected] (E. Haramoto).
groups consisting of more than 30 genotypes (Heller et al., 2001; Muljono et al., 2001; Okamoto et al., 1999, 2001; Peng et al., 2002). TTV is prevalent worldwide in general populations, such as blood donors, as well as in patients of hepatitis (Okamoto et al., 1998a; Biagini, 2001; Iriyama, 1999; Kato, 2000; Niel, 1999; Takahashi et al., 1998). TTV was originally suspected to cause hepatitis, but recent studies have indicated that it may not cause serious health problems (Abe et al., 1999). However, several genotypes of TTV are thought to be responsible for human diseases (Takahashi et al., 1998; Umemura et al., 2001). TTV is excreted in feces as a complete virion, suggesting that the fecal-oral route is a predominant mode of its transmission (Okamoto et al., 1998b).
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The TaqMan PCR quantification system has been developed and applied to the detection of TTV by many researchers (Iriyama et al., 1999; Kato et al., 2000; Takahashi et al., 1998; Moen et al., 2002; Tokita et al., 2002). Primers based on the sequence of a coding region can be used to detect TTV of group 1, while TTV of all known groups can be detected using primers based on the sequence of a non-coding region (Okamoto et al., 1998b, 2000). In order to clarify the infection route of TTV, it is important to accumulate the information on the occurrence of TTV in the environment. TTV has been successfully detected in 13% (8/63) and 2% (1/48) of 40 ml influent and effluent samples from a wastewater treatment plant (WWTP) in India, respectively (Vaidya et al., 2002). It has been also isolated from 8% (9/113) of shellfish samples in Norway (Myrmel et al., 2004). In our previous study, 3 (5%) of 64 river water samples in Japan showed positive for TTV (Haramoto et al., 2005). In this study, four kinds of wastewater samples (influent, secondary effluent, final effluent, and effluent for reuse) were collected monthly from eight WWTPs in Japan for 1 year, from July 2003 to June 2004, and applied to the detection of TTV by using TaqMan PCR. The concentration of total coliform (TC), which is currently used as an indicator microorganism in the effluents from WWTPs in Japan, was also tested, and the relationship between TC and TTV was determined.
2. Materials and methods 2.1. Collection of wastewater samples Eight WWTPs (named WWTPs A to H) were selected from seven prefectures in Japan: two WWTPs in Tokyo prefecture (WWTPs A and B), and one WWTP each in Kanagawa (WWTP C), Saitama (WWTP D), Ibaraki (WWTP E), Osaka (WWTP F), Kyoto (WWTP G), and Shiga (WWTP H) prefectures. These WWTPs treat 1.0 107 to 2.1 108 m3 of sewage per year with an average of 5.8 107 m3 per year. A standard activated sludge process is installed at WWTPs A, B, D, F, and G. Meanwhile, an anaerobic-oxic (AO) or an anaerobicanoxic-oxic (A2O) activated sludge process is installed at WWTP C or H, respectively. WWTP E is equipped with a nitrifying-denitrifying activated sludge process. At each WWTP, influent samples, secondary effluent samples before chlorination, and final effluent samples after chlorination were collected once a month for 1 year, from July 2003 to June 2004. In order to obtain sufficient contact time with chlorine, the final effluent samples were kept at room temperature for 15 min after the sampling, followed by the addition of sodium thiosulfates for the neutralization of chlorine. In total,
2009
96 influent, 96 secondary effluent, and 95 final effluent samples were analyzed. Due to the failure of the sampling, the final effluent sample from WWTP H was not collected in October 2003. At WWTPs B and C, effluent samples for reuse, treated with sand filtration and ozonation, were also collected in addition to the other samples. All samples were collected and stored in plastic bottles on ice. The samples were delivered to the laboratory within 1 day after collection and analyzed immediately upon arrival. All the samples were assayed for TC by a single agar layer method using chromocult coliform agar (Merck, Tokyo, Japan) according to the protocols described by the manufacture. Suspended solid (SS) was also measured following the standard methods of the examination of water in Japan (Japan Water Works Associtation, 1993). Temperature and pH of the samples were determined on site upon collection. 2.2. Concentration of wastewater samples The method used for the concentration of wastewater samples was described previously (Katayama et al., 2002). Briefly, 2.5 M MgCl2 was inoculated into the sample to obtain a concentration of 25 mM, then the sample was passed through an HA filter (0.45 mm pore size and 90 mm diameter; Millipore, Tokyo, Japan) attached to a glass filter holder (Advantec, Tokyo, Japan). The volumes filtered were 100 ml for influent, and 1000 ml for secondary effluent, final effluent, and effluent for reuse. The filter was rinsed with 200 ml of 0.5 mM H2SO4 (pH 3.0), followed by elution of viruses with 10 ml of 1 mM NaOH (pH 10.8). The filtrate was recovered in a tube containing 50 ml of 100 mM H2SO4 (pH 1.0) and 100 ml of 100 TrisEDTA buffer (pH 8.0) for neutralization, followed by ultrafiltration using a Centriprep YM-50 (Millipore) at 2500 rpm for 10 min. The sample which did not pass through an ultrafiltration membrane of the Centriprep YM-50 (approximately 2 ml) was applied to further centrifugation at 2500 rpm for 5 min to obtain a final volume of 700 ml. 2.3. DNA extraction and TaqMan PCR Viral DNA was extracted from 200 ml of the final concentrated sample using a QIAamp DNA mini kit (Qiagen, Tokyo, Japan) according to the protocols described by the manufacture. Five microliters out of 200 ml of the resulting extracted DNA was mixed with 45 ml of a reaction buffer containing 25 ml of 2 TaqMan universal PCR master mix (Applied Biosystems, Tokyo, Japan), 2 ml of 10 mM sense primer (50 -CGGGTGCCGDAGGTGAGTTTACAC-30 ), 2 ml of 10 mM antisense primer (50 -GAGCCTTGCCCATRGCCCGGCCAG-30 ), and 3 ml of 5 mM TaqMan probe
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2010
(50 -FAM [6-carboxyfluorescein]-AGTCAAGGGGCAATTCGGGCTCGGGA-TAMRA [6-carboxytetramethylrhodamine]-30 ) (Tokita et al., 2002), where R is A or G; and D is A, G, or T. Subsequently, the mixture was added to a well of a 96 well micro plate (Applied Biosystems). The plate was incubated at 50 1C for 2 min and 95 1C for 10 min, followed by 50 cycles at 95 1C for 10 s and at 62 1C for 30 s, and it was finally cooled to 4 1C. The DNA amplification was determined using the ABI PRISM 7000 sequence detection system (Applied Biosystems). Three wells were adopted for the detection of TTV for one sample. A positive result was obtained when at least one of the three wells showed the DNA amplification. Two hundred microliters out of 700 ml of the final concentrated sample was applied to DNA extraction, and 15 ml out of 200 ml of the extracted DNA was used for the detection of TTV. Therefore, volumes tested for the presence of TTV were equivalent to approximately 2.1 ml for influent, and 21 ml for secondary effluent, final effluent, and effluent for reuse.
3. Results 3.1. Influent samples Table 1 shows the water quality parameters of wastewater samples. Temperatures of the samples collected in winter were usually lower than 20 1C, while those collected in summer sometimes exceeded 25 1C. The concentration of TC in the influent ranged from 80,000 to 4,100,000 CFU/ml, but the monthly mean values of TC did not show any seasonal variation. As summarized in Table 2, TTV was detected in 93 of 96 influent samples, positive for 97%. 3.2. Secondary and final effluent samples Secondary and final effluent samples were collected before and after chlorination, respectively. The annual mean values of TC were 680 CFU/ml (range: 0–5400 CFU/ml) and 130 CFU/ml (range: 6.5–2800 CFU/ml) for the secondary and final effluent
Table 1 Water quality of wastewater samples Sample type (no. of samples)a
Influent (96) Secondary effluent (96) Final effluent (95) Effluent for reuse (24)
Mean (range) Temperature (1C)
pH
21 22 22 22
7.4 6.7 6.7 7.1
(14–28) (15–28) (15–29) (17–27)
(6.6–8.4) (6.3–7.7) (6.3–7.4) (6.7–7.5)
SS (mg/l)
TC (CFU/ml)
190 (67–560) 1.9 (0–6.5) 1.9 (0–12) 2.8 (0–23)
360,000 (80,000–4,100,000) 680 (0–5,400) 130 (6.5–2,800) 6.0 (0–54)
a Influent, secondary effluent, and final effluent samples were collected from eight WWTPs, and effluent for reuse were collected from two of eight WWTPs.
Table 2 Detection of TTV in wastewater samples Time of collection (year and month)
2003
2004
Total (% positive)
July Aug. Sep. Oct. Nov. Dec. Jan. Feb. Mar. Apr. May June
No. of positive samples/no. of samples tested Influent
Secondary effluent
Final effluent
Effluent for reuse
8/8 8/8 8/8 7/8 8/8 7/8 8/8 7/8 8/8 8/8 8/8 8/8
2/8 2/8 0/8 2/8 2/8 2/8 0/8 0/8 1/8 2/8 3/8 1/8
3/8 3/8 3/8 2/7 2/8 0/8 2/8 3/8 1/8 1/8 2/8 1/8
0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2
93/96 (97%)
17/96 (18%)
23/95 (24%)
0/24 (0%)
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3.3. Effluent samples for reuse Effluents for reuse were collected from two (WWTPs B and C) out of eight WWTPs. The advanced wastewater treatment process using sand filtration followed by ozonation did not remove SS effectively but reduced TC more than 1.4 log (Table 1). All 24 effluents for reuse were negative for TTV, while it was detected in 6 (25%) of 24 final effluents collected from WWTPs B and C (Table 2). 3.4. Difference among WWTPs The difference of the TC concentrations in influent and final effluent samples among eight WWTPs are shown in Fig. 1. The annual mean concentration of TC in the influent was similar for all WWTPs, ranging from 210,000 (WWTP G) to 560,000 CFU/ml (WWTP B). The annual mean concentration of TC in the final effluent collected from each WWTP ranged from 23 (WWTP H) to 460 CFU/ml (WWTP C). Out of 12 final effluent samples collected from each WWTP, TTV was detected twice (17%), three times (25%), and four times (33%) at two (WWTPs A and B), one (WWTP E), and four (WWTPs C, D, F, and G) WWTPs, respectively. However, all 11 final effluent samples collected from WWTP H were negative for TTV.
4. Discussion TTV is an emerging virus which was discovered as an agent of unknown hepatitis in Japan in 1997. TTV is isolated not only in the patients of hepatitis but also in the healthy persons (Okamoto et al., 1998a; Biagini et al., 2001; Iriyama et al., 1999; Kato et al., 2000; Niel et al., 1999; Takahashi et al., 1998), thus it is strongly
Influent 7
Final effluent
6 TC (log CFU/mL)
samples, respectively (Table 1). The annual mean inactivation ratio of TC by chlorination was 0.71 log with a maximum value of 2.2 log. On the other hand, chlorination did not affect the results of PCR for TTV. TTV was detected in 18% (17/96) and 24% (23/95) of secondary and final effluent samples, respectively (Table 2). Comparison between the influent and effluent samples revealed that 89–100% of SS were removed by the wastewater treatment. The total reduction ratio of TC throughout the treatment ranged from 2.0 to 5.0 log with an average of 3.4 log. The mean concentration of TC was 230 CFU/ml (range: 20–2800 CFU/ml) for TTVpositive final effluent samples, while it was 140 CFU/ml (range: 6.5–1400 CFU/ml) for TTV-negative final effluent samples.
2011
5 4 3 2 1 0
A
B
C
D
E
F
G
H
WWTP Fig. 1. Difference of TC concentrations among eight WWTPs. The plot, upper bar, and lower bar show the geometric mean, maximum, and minimum data of TC concentrations in 11 or 12 the samples collected for 1 year, respectively.
suspected to be transmitted by the fecal-oral route (Okamoto et al., 1998b). There are only a few studies on the detection of TTV in the environmental samples: influent and effluent samples from a WWTP in India (Vaidya et al., 2002), shellfish samples in Norway (Myrmel et al., 2004), and river water samples in Japan (Haramoto et al., 2005). In this study, the occurrence of TTV in wastewater samples collected from eight WWTPs in Japan was surveyed for 1 year. The samples were concentrated using the method developed in our laboratory (Katayama et al., 2002), followed by TaqMan PCR using the primer pairs and the TaqMan probe specific for TTV of all known genotypes (Tokita et at., 2002). In total, TTV was detected in 97% (93/96), 18% (17/96), or 24% (23/ 95) of influent, secondary effluent, or final effluent samples, respectively, while no TTV was detected in 24 effluent samples for reuse (Table 2). The presence or absence of TTV was tested by using only small volumes which were equivalent to approximately 2.1 ml (influent) or 21 ml (secondary effluent, final effluent, and effluent for reuse) of the original samples. Compared with the study conducted by Vaidya et al. in India (Vaidya et al., 2002), this study showed a very high positive ratio of TTV from influent samples. This might be explained by the fact that TTV was isolated in nearly 90% of blood donors in Japan (Kato et al., 2000; Takahashi et al., 1998). Moreover, the high positive ratio of TTV observed in this study might also be attributable to our detection method which possibly has an advantage at the detection of TTV including the high recovery efficiency in the concentration process, and/or the high sensitivity of the TaqMan PCR system. TC was also tested in this study because it is an indicator microorganism in the standard for the
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discharge from a WWTP in Japan: the concentration of TC must be lower than 3000 CFU/ml. The concentrations of TC in wastewater samples were stable throughout the year. That is, the mean values of the concentrations of TC were 360,000, 680, 130, and 6.0 CFU/ml for influent, secondary effluent, final effluent, and effluent for reuse, respectively (Table 1). All 95 final effluent samples met the standard for TC. Chlorination achieved a 0.71 log reduction of TC on the average, but the positive ratio of TTV was not decreased by chlorination (Table 2). There is no data on the tolerance of TTV to chlorine, but it is known that the genomes of enteric viruses, such as poliovirus and hepatitis A virus, inactivated by chlorine can be detected by PCR without any specific treatment (Nuanualsuwan and Cliver, 2002). Therefore, the PCR test adopted in this study may have detected the DNA of chlorine-inactivated TTV. Further studies are required to evaluate the resistance of TTV to chlorine. There was no significant difference between the concentration of TC in TTV-positive final effluents and that in TTV-negative final effluents (t-test, P40.05), implying a possibility that TC may not indicate the fate of TTV discharged from WWTPs to the water environment. The annual mean reduction ratio of TC at WWTP H was 4.3 log, while those at other seven WWTPs ranged from 2.8 to 3.8 log (Fig. 1). TTV was not detected in effluent samples collected from WWTP H but detected in 17–33% of effluent samples collected from other seven WWTPs. These results suggest that the A2O activated sludge process conducted at WWTP H is an effective system to reduce the concentration of bacteria and viruses, including TC and TTV, in the influent. The advanced wastewater treatment using sand filtration and ozonation was also considered as a proper method to remove them. Monitoring of viruses in the influent of a WWTP provides useful information to determine their actual incidence in its service area. From the results in this study, it is suggested that TTV is epidemic in Japan throughout the year and that TTV is continuously discharged into the water environment after the treatment at WWTPs. The fate of TTV discharged from WWTPs need to be studied in the future.
5. Conclusion TTV, an emerging hepatitis virus, was detected in 97% (93/96), 18% (17/96), 24% (23/95), and 0% (0/24) of influents, secondary effluents, final effluents, and effluents for reuse collected from eight WWTPs in Japan, respectively. No clear correlation was observed between TTV and TC in the final effluents, implying the need for further studies on the appropriate indicators of
TTV. The high incidence of TTV in the influents indicated that the virus is epidemic widely in Japan throughout the year.
Acknowledgements This study was supported by the Microbial Water Quality Project on Treated Sewage and Reclaimed Wastewater, Ministry of Land, Infrastructure and Transport Government of Japan. We also thank Mr. Atsushi Tajima and Mr. Kensuke Sakurai (National Institute for Land and Infrastructure Management, Ibaraki, Japan), and Ms. Hozue Kuroda (University of Tokyo, Tokyo, Japan), for their help.
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