Occurrence of Viruses and Protozoa in Drinking Water Sources of ...

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September 2012 , Volume 4, Issue 3, pp 93–101 ... Haramoto, E., Kitajima, M., Kishida, N. et al. Food Environ Virol (2012) 4: 93. doi:10.1007/s12560-012-9082-0.
Food Environ Virol (2012) 4:93–101 DOI 10.1007/s12560-012-9082-0

ORIGINAL PAPER

Occurrence of Viruses and Protozoa in Drinking Water Sources of Japan and Their Relationship to Indicator Microorganisms Eiji Haramoto • Masaaki Kitajima • Naohiro Kishida • Hiroyuki Katayama Mari Asami • Michihiro Akiba



Received: 28 May 2012 / Accepted: 12 June 2012 / Published online: 4 July 2012 Ó Springer Science+Business Media, LLC 2012

Abstract A nationwide survey of viruses, protozoa, and indicator microorganisms in drinking water sources of Japan was conducted. Among 64 surface water samples collected from 16 drinking water treatment plants, 51 (80 %) samples were positive for at least one of the 11 pathogen types tested, including noroviruses of genogroups I (positive rate, 13 %) and II (2 %), human sapoviruses (5 %), human adenoviruses of serotypes 40 and 41 (39 %), Cryptosporidium oocysts (41 %), and Giardia cysts (36 %). Total coliforms, Escherichia coli, and F-specific E. Haramoto (&) International Research Center for River Basin Environment, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan e-mail: [email protected] M. Kitajima Department of Soil, Water and Environmental Science, College of Agriculture and Life Sciences, The University of Arizona, 1117 E. Lowell Street, Tucson, AZ 85721, USA e-mail: [email protected] N. Kishida  M. Asami  M. Akiba Division of Water Management, Department of Environmental Health, National Institute of Public Health, 2-3-6 Minami, Wako, Saitama 351-0197, Japan e-mail: [email protected] M. Asami e-mail: [email protected] M. Akiba e-mail: [email protected] H. Katayama Department of Urban Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan e-mail: [email protected]

coliphages were detected in 63 (98 %), 33 (52 %), and 17 (27 %) samples, respectively, and E. coli was judged to be the most suitable indicator of pathogen contamination of drinking water sources. Genogroup-specific real-time PCR for F-specific coliphages revealed the presence of F-specific RNA coliphages of animal genogroup I and human genogroups II and III in 13 (41 %), 12 (39 %), and 1 (3 %), respectively, of 31 plaques isolated. Keywords Drinking water source  Indicator microorganism  Protozoa  Public health  Virus

Introduction Many rivers with intakes for drinking water treatment plants (DWTPs) exist downstream of wastewater treatment plants (WWTPs) and/or septic tanks. Therefore, pathogen contamination of surface water, a major source for drinking water production, is a serious public health problem that can affect the entire population living in the DTWP-supplied areas. Moreover, animal fecal contamination of surface water can pose the risk of pathogen infection to humans because many types of waterborne pathogens are also zoonotic agents (Plutzer et al. 2010; Smith and Nichols 2010). Numerous outbreaks of waterborne diseases because of contaminated drinking water have been reported worldwide (Fong and Lipp 2005; Plutzer et al. 2010; Smith and Nichols 2010). The largest waterborne outbreak in recent years occurred in Milwaukee, Wisconsin, USA, in 1993, where 403,000 people fell ill with cryptosporidiosis (MacKenzie et al. 1994). In Japan, the most notorious waterborne outbreak occurred in Ogose, Saitama Prefecture, in June 1996, where 8,812 people, or 71.4 % of the

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town population, were infected with Cryptosporidium oocysts by drinking contaminated tap water (Yamamoto et al. 2000). A waterborne outbreak of norovirus (NoV)associated gastroenteritis was also reported in a small village in Akita Prefecture in March 2005, where 29 people were infected with NoVs of genogroup II (GII) by drinking contaminated well water supplied without chlorination (Saito 2005). In these outbreaks, increased concentrations of pathogens in raw water are well documented, as are the unexpected decreases in the efficiency of pathogen removal/disinfection during drinking water treatment. In Japan, following the outbreak of cryptosporidiosis in Ogose, the occurrence of Cryptosporidium oocysts and Giardia cysts in river water used for drinking water production was surveyed, showing high positive detection rates in the samples (Hashimoto et al. 2002; Kishida et al. 2011). Meanwhile, various viruses such as NoVs and human sapoviruses (HuSaVs) were frequently detected in surface water samples of the Tamagawa River, which is a major river that flows through the Tokyo metropolitan area (Haramoto et al. 2005, 2008; Kitajima et al. 2009, 2010a, b). These viruses were more abundant in the midstream and downstream areas of the river, where the effluents from WWTPs account for nearly half of the river water. Surface water from the upstream area, with low levels of viral contamination, is used as the raw water for drinking water, whereas not surface water but subsoil water is utilized to produce drinking water from the midstream and downstream areas; therefore, high levels of viral contamination of the surface water in the midstream and downstream areas may not be directly related to the risk of viral infection because of drinking water. These previous studies were aimed at determining the occurrence of viruses or protozoa in certain river basins (Haramoto et al. 2005, 2008; Hashimoto et al. 2002; Kishida et al. 2011; Kitajima et al. 2009, 2010a, b), but almost no data are available about the pathogen occurrence in many other regions of Japan. A nationwide survey of various waterborne pathogens in drinking water sources needs to be performed to understand the actual occurrence of the pathogens in the whole country and to evaluate the risk of pathogen infection to humans because of contaminated drinking water. Meanwhile, routine monitoring of pathogens in drinking water sources is sometimes time consuming and not cost effective compared with the analysis of conventional indicator microorganisms. Thus, it is also important to evaluate the relationships between the occurrence of pathogens and indicator microorganisms. To the best of our knowledge, no studies on the pathogen occurrence targeting both viruses and protozoa and on their relationships to indicator microorganisms in drinking water sources have been reported. Given this background, the aims of this study were to determine the overall occurrence of viruses and protozoa in drinking water sources of Japan and to evaluate the

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relationships between the occurrence of pathogens and indicator microorganisms. Sixty-four raw water samples were collected from 16 DWTPs between 2008 and 2009. Each year, water sampling was conducted in July and in December to be representative of the summer and winter seasons, respectively. Viruses and protozoa were concentrated by a method for simultaneous concentration of viruses and protozoa from single samples using electronegative membranes, followed by detection of a total of 11 types of the pathogens using real-time PCR or fluorescence microscopy. RNA viruses, NoVs GI and GII, and HuSaVs were tested because they are important causes of acute viral gastroenteritis worldwide. Bovine-specific NoVs GIII and emerging NoVs GIV were also tested. In addition to the well-studied human adenoviruses of serotypes 40 and 41 (HuAdVs 40/41), emerging viruses such as torque teno viruses (TTVs), and JC and BK polyomaviruses (JCPyVs and BKPyVs) were tested as DNA viruses. Cryptosporidium oocysts and Giardia cysts were the protozoa tested. Total coliforms, Escherichia coli, and F-specific coliphages were tested as indicator microorganisms. Furthermore, considering the possibility that the raw water samples were contaminated with both human and animal feces, F-specific coliphage plaques isolated from the samples were subjected to genogroup-specific real-time PCR for four genogroups of F-specific RNA (F-RNA) coliphages to differentiate between human and animal fecal contamination of the drinking water sources. The genogrouping using F-RNA coliphages is based on the knowledge that genogroup II (GII) and GIII F-RNA coliphages are generally derived from human feces, whereas GI and GIV are derived from animal feces (Vinje´ et al. 2004).

Methods Collection of Water Samples A total of 16 DWTPs (A–P) were selected based on their locations adjacent to major water systems (class A rivers) in Japan and the fact that their surface water was used as raw water for drinking water production. As shown in Table 1, at least two DWTPs were selected from each of the six regions of Japan, each comprising 7–9 prefectures. Total river flows in the water systems where the studied DWTPs are located account for *40 % of those in the class A rivers all over Japan. Most of these DWTPs are located downstream of one or more WWTPs and/or septic tanks. In addition, for some DWTPs, there are many livestock production facilities in the upstream areas of the river basins, implying a possible source of animal fecal contamination of surface water used for drinking water production.

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Table 1 DWTPs studied in this study Region

Hokkaido– Tohoku

Population (in thousands)

No. of prefectures

No. of DWTPs studied (ID)

No. of samples collected

14,877

7

4 (A–D)

16

Kanto

42,053

7

3 (E–G)

12

Chubu

21,774

9

2 (H–I)

8

Kansai

22,684

7

2 (J–K)

8

Chugoku– Shikoku

11,559

9

3 (L–N)

12

Kyushu– Okinawa Total

14,566

8

2 (O–P)

8

127,510

47

16

64

Water sampling was conducted four times in July (i.e., summer season) and December (i.e., winter season) of both 2008 and 2009. At each sampling time, raw surface water samples ([2.2 l each) were collected from these DWTPs, stored in plastic or glass bottles on ice, and delivered to the laboratory of the National Institute of Public Health, Wako, Japan, within 2 days of sample collection. pH values of these samples ranged from 6.3 to 7.8, with a mean value of 7.1, whereas turbidity ranged from 1 to 32, with a mean value of 7.3. The sampling was normally conducted in the morning on sunny or cloudy days, except for a few samples that were collected during small rainfall events due to the limited availability of schedules. Some samples were also collected within a few days after rainfall events. Therefore, the occurrence of pathogens and indicator microorganisms in several samples might have been affected by rainfall as reported in previous studies (Surbeck et al. 2006; Rijal et al. 2009). Concentration of Viruses and Protozoa Viruses in the raw water samples were concentrated with a previously developed method using an electronegative membrane (Katayama et al. 2002). In brief, the raw water samples (2 l each) were mixed with 20 ml of 2.5 mol/l MgCl2, followed by filtration through a mixed cellulose ester membrane (pore size, 0.45 lm; diameter, 90 mm; Millipore, Billerica, USA) attached to a glass membrane holder (Advantec, Tokyo, Japan) to adsorb viruses to the membrane using electrostatic interactions between the viruses and the electronegative membrane in the presence of cations and to trap protozoa physically to the membrane. For several samples, the filtered volumes were smaller than 2 l (1.0–1.6 l) because of observed membrane clogging. Subsequently, 200 ml of 0.5 mmol/l H2SO4 (pH 3) was passed through the membrane to remove magnesium ions and other electropositive substances, followed by filtration of

10 ml of 1 mmol/l NaOH (pH 11) for elution of viruses from the membrane. The filtrate was recovered in a 50-ml plastic tube containing 50 ll of 0.1 mol/l H2SO4 (pH 1) for neutralization, which was further concentrated using the Centriprep YM-50 device (Millipore) to a virus-concentrated sample with a volume of 0.50–0.90 ml (mean, 0.59 ml). The samples were stored at -20 °C until further analysis. After viral elution, the membrane was subsequently used to recover protozoan (oo)cysts. In brief, the membranes were detached from their holder, followed by vigorous vortexing in the presence of a ball-shaped stirring bar and 10 ml of an elution buffer containing 0.2 g/l Na4P2O710H2O, 0.3 g/l C10H13N2O8Na33H2O, and 0.1 ml/l Tween 80 in a 50-ml plastic tube. The water portions of the samples were recovered as the protozoa-concentrated sample. Detection of Viral Genomes Viral DNA was extracted from 200 ll of the virus-concentrated samples using the QIAamp DNA Mini Kit (Qiagen, Valencia, USA). Viral RNA was also extracted from 140 ll of the samples using the QIAamp Viral RNA Mini Kit (Qiagen), followed by reverse transcription (RT) using the High Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, USA). In brief, 16 ll of the extracted RNA was added to 16 ll of an RT mixture containing 3.2 ll of 109 RT buffer, 3.2 ll of 109 RT random primers, 1.6 ll of 20 units/ll RNase inhibitor, 1.6 ll of 50 units/ll MultiScribe reverse transcriptase, 1.28 ll of 100 mmol/l dNTP Mix, and 5.12 ll of PCRgrade water, followed by incubation at 25 °C for 10 min, 37 °C for 120 min, and 85 °C for 5 min. Five microliters of each DNA/cDNA was mixed with 15 ll of a PCR mixture containing 4 ll of 59 LightCycler FastStart DNA MasterPLUS HybProbe Master Mix (Roche Diagnostics, Basel, Switzerland), 1 ll of 10 pmol/ll of both forward and reverse primers, 0.8 ll of 5 pmol/ll TaqMan or TaqMan MGB probe, and 8.2 ll of PCR-grade water. The primers and the TaqMan/TaqMan MGB probes used here were designed from the conserved nucleotide sequences of each target virus: the junction of the open reading frames (ORFs) 1 and 2 for NoVs GI–GIV (Kageyama et al. 2003; Trujillo et al. 2006; Wolf et al. 2007), the ORF1 region for HuSaVs (Haramoto et al. 2008), the fiber gene region for HuAdVs 40/41 (Ko et al. 2005), the non-coding region for TTVs (Tokita et al. 2002), and the early region for JCPyVs and BKPyVs (Pal et al. 2006). Glass capillaries containing the mixtures were placed into the LightCycler 2.0 (Roche Diagnostics) and incubated at 95 °C for 10 min, followed by 50 cycles of 95 °C for 10 s and 55–62 °C, depending on virus type, for 30 s. A separate glass capillary was used for each of the tested viruses. Tenfold serial dilutions of the recombinant plasmid

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DNA or chemically synthesized oligo-DNA were used to make a standard curve. Threshold cycle (Ct) values were determined automatically with the LightCycler software using the second derivative maximum method, followed by determination of the concentrations of the viral genomes using the standard curves. Negative controls were always included in PCR runs. In order to verify that negative PCR results were not due to the presence of PCR-inhibitory substances in the cDNA sample, 5 ll of each cDNA was mixed with known amount (1,000 copies/reaction) of internal control DNA, the chemically synthesized oligo-DNA of TTV non-coding region, followed by real-time PCR quantification as described above (Tokita et al. 2002). The TTV non-coding region sequences with a length of 79 bp were used as internal control for RNA viruses because the sequences were considered absent in cDNA samples. The PCR amplification efficiency was calculated as the ratio of total number of the internal DNA copies in the cDNA sample to that in the control sample containing no cDNA. Detection of Protozoan (Oo)cysts The protozoa-concentrated samples were subjected to immunomagnetic separation (IMS) using the Dynabeads GC-Combo (Life Technologies) according to the manufacturer’s instructions. Subsequently, the IMS-treated samples were passed through a hydrophilic polytetrafluoroethylene (PTFE) membrane (pore size, 1.0 lm; diameter, 25 mm; Advantec), followed by fluorescent staining of the protozoan (oo)cysts on the membrane using the EasyStain (BTF, North Ryde, Australia) and 40 ,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, USA). A FluoView FV1000 laser scanning confocal microscope (Olympus, Tokyo, Japan) was used to count the numbers of Cryptosporidium oocysts (round-shaped with a diameter of 4–6 lm) and Giardia cysts (oval-shaped with a diameter of 5–8 lm and a width of 8–12 lm).

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Genogrouping of F-Specific Coliphage Plaques For samples positive for F-specific coliphages, all the phage plaques that formed on the agar plates were isolated using a sterile loop and inoculated in a 1.5-ml microtube containing 500 ll of phosphate buffered saline. The microtube was vortexed vigorously, and 13 ll of the phage suspension was subjected to RNA extraction by heating at 95 °C for 5 min using the GeneAmp PCR system 9700 (Life Technologies). Then, the extracted RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit, followed by TaqMan MGB-based real-time PCR for each genogroup of F-RNA coliphages using the LightCycler 2.0. The primer pairs and TaqMan MGB probes used here were designed from 4-, 5-, 3-, and 3-phage nucleotide sequences from the GenBank database for GI, GII, GIII, and GIV F-RNA coliphages, respectively (Ogorzaly and Gantzer 2006, 2007). The concentrations of each component in the RT and PCR mixtures were the same with those used for RNA viruses. The thermal conditions were as follows: 95 °C for 10 s, and 50 cycles of 95 °C for 10 s, and 60 °C for 30 s. One glass capillary was used for each of the four genogroups, and Ct values were determined to check the genome amplification. In cases wherein a phage plaque was negative for all F-RNA coliphage genogroups, the plaque was further subjected to SYBR Green-based real-time PCR for F-specific DNA (F-DNA) coliphages (Haramoto et al. 2009b). In brief, 5 ll of heat-released DNA was mixed with 15 ll of a PCR mixture containing 4 ll of 59 LightCycler FastStart DNA MasterPLUS SYBR Green I Master Mix (Roche Diagnostics), 1 ll of 10 pmol/ll of both forward and reverse primers specific for all F-DNA coliphage genogroups (Vinje´ et al. 2004), and 9 ll of PCR-grade water. The thermal conditions were as follows: 95 °C for 10 min; and 50 cycles of 95 °C for 10 s, 50 °C for 5 s, and 72 °C for 25 s. At the end of 50 PCR amplification cycles, a melting curve analysis was performed, and a single melting temperature (Tm) peak was observed at *80 °C, which was derived from the amplified DNA of F-DNA coliphages. One glass capillary was used for each sample.

Detection of Indicator Microorganisms Statistical Analysis The concentrations of total coliforms and E. coli in 0.1 or 1 ml of the raw water samples were determined by a single agar layer method using chromocult coliform agar (Merck Chemicals, Darmstadt, Germany). After incubation at 37 °C for 24 h, blue colonies were counted as E. coli, whereas both blue and red colonies were counted as total coliforms. F-specific coliphages in 5 ml of the samples were also measured by plaque assay using the host strain Salmonella enterica serovar Typhimurium WG49 in accordance with ISO standard 10705-1 (Anonymous 1995). Phage plaques were counted after incubation at 37 °C for 24 h. A duplicate was tested for each sample.

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Statistical analysis was performed by v2 test using Microsoft Excel 2010 (Microsoft, Redmond, USA). Significance level (P value) was set at 0.05.

Results and Discussion Occurrence of RNA Viruses in Drinking Water Sources The results of the detection of viral genomes and protozoan (oo)cysts are summarized in Table 2. NoVs GI were

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Table 2 Detection of viruses and protozoa in drinking water sources of Japan Pathogen

No. of positive samples

No. of samples testing positive/total no. of samples tested (% positive)

Concentration in positive samples (min–max) (copies/l or (oo)cysts/l)

July 2008

December 2008

July 2009

December 2009

NoV GI

0

0

7

1

8/64 (13)

NoV GII NoV GIII

0 0

1 0

0 0

0 0

1/64 (2) 0/64 (0)

NoV GIV

0

0

0

0

0/64 (0)

HuSaV

0

3

0

0

3/64 (5)

HuAdV 40/41

8

6

4

7

25/64 (39)

TTV

1

0

1

0

2/64 (3)

770–920

JCPyV

1

0

1

0

2/64 (3)

290–1,300

BKPyV

0

1

0

0

1/64 (2)

250

Cryptosporidium

8

7

6

5

26/64 (41)

0.5–30

Giardia

5

8

3

7

23/64 (36)

0.5–3.8

RNA virus 270–33,000 340

460–820

DNA virus 210–17,000

Protozoa

detected in seven samples collected in July 2009 and 1 sample in December 2009, with an overall positive detection rate of 13 %. NoVs GII were detected in only 1 sample collected in December 2008, with a concentration of 340 copies/l, whereas neither bovine NoVs GIII nor emerging NoVs GIV were detected in any samples. The sample collected from DWTP A in July 2009 contained NoVs GI with a very high concentration of 33,000 copies/l. Numerous studies have demonstrated the high occurrence of NoVs GI and GII in river water worldwide (Haramoto et al. 2005; Lodder and de Roda Husman 2005; Hamza et al. 2009; Kitajima et al. 2010b). In Japan, several studies have shown the remarkable increase in concentrations of NoVs GI and GII in river water during the epidemic period (i.e., winter/early spring, between November and March), together with the emerging genogroup GIV (Haramoto et al. 2005; Kitajima et al. 2009). Interestingly, NoVs GI were found in seven of the 16 samples collected in July 2009, where only 29 cases of NoV infection all over Japan were reported to the National Institute of Infectious Diseases, Tokyo, Japan (http://idsc.nih.go.jp/iasr/index.html). Considering the relatively low Ct values (range, 34.2–39.3; corresponding to 15–370 copies/reaction), these NoV GIpositive samples were not considered as false-positive cases. Unreported NoV infections with mild symptoms may have affected the occurrence of NoVs GI in the drinking water sources. HuSaVs were detected in three samples collected in December 2008, in the epidemic period of HuSaVs in Japan, with a concentration of 460–820 copies/l. Compared with NoVs, very limited data are available about the occurrence of HuSaVs in aquatic environments. Considering the high reported occurrence of HuSaVs in river

water (Haramoto et al. 2008; Kitajima et al. 2010a; Sano et al. 2011), more studies should focus not only on qualitative/quantitative detection of HuSaVs but also on the genotyping of HuSaVs in river water to evaluate the risk of infection to humans because of contaminated drinking water or during recreational activities. Following the unexpected low positive detection rates of NoVs and HuSaVs, the PCR inhibitory effects were examined using the internal control DNA. The observed concentrations of the internal control DNA in the cDNA samples ranged from 500 to 2,330 copies/reaction (mean, 1,240 copies/reaction; n = 64), resulting in the mean PCR amplification efficiency of 124 %. These results suggest that no significant PCR inhibition was observed in all the samples tested. Occurrence of DNA Viruses in Drinking Water Sources Of all the viruses tested, HuAdVs 40/41 were detected most frequently in the water samples tested: 4–8 samples were positive for these viruses during each sampling period, showing an overall positive detection rate of 39 %. The concentrations of HuAdVs 40/41 ranged from 210 to 17,000 copies/l, with a mean concentration of 2,200 copies/ l. HuAdVs are presently proposed to be an indicator of virus contamination of the environment because of its high occurrence in aquatic environments worldwide (Pina et al. 1998); our data agreed with this study. TTVs, JCPyVs, and BKPyVs were also expected to be appropriate indicators for viral contamination of drinking water sources because of their excretion in the feces or urine of healthy individuals (Cole and Conzen 2001; Irshad et al. 2006). However, compared with HuAdVs 40/41,

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these viruses were detected less frequently in the raw water samples: only two, two, and one samples were positive for TTVs, JCPyVs, and BKPyVs, respectively. The low occurrence of TTVs in river water is also documented in a previous study conducted in the Tamagawa River basin, Tokyo, Japan (Haramoto et al. 2005). These results suggest that TTVs, JCPyVs, and BKPyVs are inadequate as indicators of viral contamination of drinking water sources. Occurrence of Protozoa in Drinking Water Sources The concentration method used in this study consists of two parts. The first part is the concentration of viruses by a previously developed method using the mixed cellulose ester membrane with a pore size of 0.45 lm (Katayama et al. 2002), which has been widely used in detecting various types of viruses in aquatic environments (Haramoto et al. 2008; Katayama et al. 2008; Kitajima et al. 2009). The second part is the recovery of protozoan (oo)cysts from the membrane by vortexing in the elution buffer. Protozoan (oo)cysts were expected to be physically trapped to the membrane along with other bacteria, but the membranes were discarded after the viral elution in the above previous studies. Our method has the advantage of concentrating both viruses and protozoa in a single water sample, with easy operations. By the concentration method combined with fluorescence microscopy analysis, Cryptosporidium oocysts and Giardia cysts were successfully detected in 26 (41 %) and 23 (36 %) of the 64 raw water samples, respectively. Both Cryptosporidium oocysts and Giardia cysts were detected in 12 samples (19 %), only Cryptosporidium oocysts were detected in 14 samples (22 %), only Giardia cysts were detected in 11 samples (17 %), and none was detected in the remaining 27 samples (42 %). The concentrations of the protozoa were mostly lower than 2.0 (oo)cysts/l. However, one sample collected from DWTP F in December 2009 contained Cryptosporidium oocysts with a very high concentration of 30 oocysts/l. The highest concentration of Giardia cysts (3.8 cysts/l) was also obtained from this sample. Conversely, the concentrations of the protozoa at this DWTP during the other three sampling periods were lower than 1.0 (oo)cyst/l. These results suggest that the sample in December 2009 was sporadically contaminated by high levels of infected human and/or animal feces. Further DNA sequencing analysis will contribute to determine the source of protozoan contamination of drinking water sources. In addition, more frequent samplings are required for better understanding of temporal variations in the concentrations of pathogens in water. By combining the results of the detection of viruses and protozoa, 51 (80 %) of the 64 water samples tested were positive for at least one of the 11 pathogen types. Of the 51

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samples, 22 samples tested positive for only one pathogen type, followed by 20 samples having tested positive for two types, seven samples tested positive for three types, and two samples tested positive for four types. By DWTPs, all the water samples collected from 8 DWTPs were always positive for at least 1 pathogen type. Five and two DWTPs were positive for at least one pathogen type in three and two of the samples, respectively, whereas DWTP P was the only plant where no pathogens were detected throughout the study. In previous studies, 10 l or larger volumes of surface water samples were sometimes processed for virus or pathogen concentration procedures, partially probably because of insufficient recoveries of pathogens by concentration methods (Lee et al. 2007; Verheyen et al. 2009). Conversely, the successful detections of various types of viruses and protozoa from much smaller volumes of water samples (i.e., up to 2 l) strongly suggest high pathogen recovery rates by the concentration method employed in this study. Actually, practical high recoveries of poliovirus were obtained from various poliovirus-inoculated environmental water samples by using this method (Katayama et al. 2008; Haramoto et al. 2009a). High recoveries of protozoan (oo)cysts from river water samples (up to 80 %) were also observed by recovery tests using the ColorSeed (BTF), which contained exactly 100 Cryptosporidium oocysts and 100 Giardia cysts stained with a Texas Red dye (unpublished data). Unfortunately, although no recovery tests were performed against the water samples collected in this study, our future surveys will include the experiments regarding not only the detection of indigenous pathogens but also the evaluation of pathogen recoveries, to determine the actual concentration of the pathogens in aquatic environments. Occurrence of Indicator Microorganisms in Drinking Water Sources The results of the detection of the indicator microorganisms are shown in Table 3. According to the Japanese standard for river water used for drinking water production, the concentration of total coliforms should not exceed 5,000 most probable number (MPN)/100 ml, corresponding to 50 colony-forming units (CFU)/ml (MOE 1971); 52 % (33/64) of the tested samples exceeded this guideline value, showing the highest concentration of 490 CFU/ml. Only one sample, which was collected from DWTP D in December 2008, was negative for total coliforms. Escherichia coli and F-specific coliphages were detected in 33 (52 %) and 17 (27 %) of the 64 samples, with the highest concentrations of 8.5 CFU/ml and 0.4 plaqueforming units (PFU)/ml, respectively. The positive detection rate of E. coli was significantly higher in summer

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Table 3 Detection of indicator microorganisms in drinking water sources of Japan Microorganism

Total coliform

No. of positive samples July 2008

December 2008

July 2009

December 2009

No. of samples testing positive/total no. of samples tested (% positive)

Concentration in positive samples (min–max) (CFU/ml or PFU/ml)

16

15

16

16

63/64 (98)

1.0–490

E. coli

9

5

15

4

33/64 (52)

0.5–8.5

F-specific coliphage

3

4

2

8

17/64 (27)

0.1–0.4

(24/32, 75 %) than in winter (9/32, 28 %; P \ 0.05). Conversely, the positive detection rate of F-specific coliphages in winter (12/32, 38 %) was higher than that in summer (5/32, 16 %; P \ 0.05). Relationship Between Pathogens and Indicator Microorganisms The relationships between the occurrence of the pathogens and the indicator microorganisms in the water samples were examined by comparing a positive detection rate of the indicator microorganisms in the pathogen-positive samples and that in the pathogen-negative samples. E. coli was detected in 32 (63 %) of 51 samples which were positive for at least one pathogen type, whereas only 1 (8 %) of 13 pathogen-negative sample tested positive for E. coli. The positive detection rate of E. coli in the pathogen-positive samples was significantly higher than that in the pathogen-negative samples (P \ 0.05). F-specific coliphages were also more frequently detected in the pathogen-positive samples (16/51, 31 %) than the pathogennegative samples (1/13, 8 %), but the difference was not significant (P [ 0.05).

Because total coliforms were detected in almost all the samples tested, the Japanese guideline value for river water used for drinking water production (50 CFU/ml) (MOE 1971) was used to divide these samples into two groups. The group with total coliform concentrations of higher than 50 CFU/ml showed a positive detection rate of 82 % (27/ 33) for the pathogens, which was not significantly different from that in the group with total coliform concentrations of lower than 50 CFU/ml (24/31, 77 %; P [ 0.05). These results suggest that the detection of E. coli is the most suitable method to predict the presence of pathogens in drinking water sources of Japan among the three indicator microorganisms tested. Genogrouping of F-Specific Coliphage Plaques Isolated from Drinking Water Sources Table 4 summarizes the results of genogrouping of F-specific coliphage plaques isolated from the water samples. Among the 16 DWTPs studied, F-specific coliphages were detected at least once at 10 DWTPs, and a total of 31 phage plaques were obtained from these samples. Positive fluorescence signals were observed in 14 (45 %), 12

Table 4 Genogrouping of F-specific coliphage plaques isolated from drinking water sources of Japan DWTP

Genogroup identified (no. of phage plaques)a July 2008

December 2008

July 2009

December 2009

A







GII (1)

B

GII (3), unc (1)

GI (1), GII (1)

GI (1)

GII (1), GIII (1), unc (1)

No. of phage plaques isolated 1 10

C







GII (1)

1

E

unc (1)

GI (1), GII (1)

unc (1)

GI (2), unc (1)

7

G







GII (2)

2

H

GI (2)

GI (4)





6

I



GI (1)





1

K







GII (1)

1

M







GI (1)

1

O







GII (1)

No. of phage plaques isolated

7

9

2

13

a

1 31

GI, GII, and GIII, F-RNA coliphages of GI, GII, and GIII, respectively

unc unclassified, – negative for F-specific coliphage

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100

(39 %), and 1 (3 %) of the 31 phage plaques by real-time PCR specific for GI, GII, and GIII F-RNA coliphages, with Ct values of 14.8–37.8, 15.4–31.5, and 16.9, respectively. One of the two phage plaques collected from DWTP G in December 2009 showed positive fluorescence signals for both GI and GII F-RNA coliphages, with Ct values of 37.8 and 26.0, respectively. This phenomenon has been documented in a previous study (Haramoto et al. 2009b): agar surrounding a target plaque may have been contaminated with a small and almost invisible plaque from another genogroup. Therefore, this phage plaque was regarded as GII considering the lower Ct value for GII. Unlike other three genogroups, no phage plaques were positive for GIV F-RNA coliphages. Moreover, five phage plaques negative for all four F-RNA coliphage genogroups were negative for F-DNA coliphages by SYBR Greenbased real-time PCR. As a result, 13 (41 %), 12 (39 %), and 1 (3 %) phage plaque were classified into GI, GII, and GIII F-RNA coliphages, respectively, whereas five phage plaques (16 %) remained unclassified. Primer pairs and TaqMan MGB probes used here were designed from a total of 15 nucleotide sequences of F-RNA coliphages available in the DNA database (Ogorzaly and Gantzer 2006, 2007). More recently, a few real-time PCR assays have been developed based on more nucleotide sequence information—19 nucleotide sequences by Wolf et al. (2008) and 30 nucleotide sequences by Friedman et al. (2011). These realtime PCR assays may contribute to characterization of the genetics of the unclassified phage plaques. On the basis of the classification of GII and GIII F-RNA coliphages as human genogroups and GI and GIV as animal genogroups (Vinje´ et al. 2004), the raw water samples at DWTPs A, C, G, K, and O were contaminated with human feces, those at DWTPs H, I, and M were contaminated with animal feces, and those at DWTPs B and E were contaminated by both human and animal feces. These results indicate that the relative contribution of human and animal fecal contamination is quite different depending on DWTP locations. In summary, this study provides important information about the overall occurrence of viruses and protozoa in drinking water sources and their relationship to indicator microorganisms. This is the first study to demonstrate the occurrence of various types of viruses and protozoa in drinking water sources of Japan. Further studies will focus on the genetic analysis of pathogens in drinking water sources to evaluate the risk of pathogen infection to humans because of contaminated drinking water. Acknowledgments This study was partially supported by the Environmental Research in Japan, ‘‘Evaluation and control of health risk from human-animal pollution sources in public water bodies,’’ from the Ministry of Environment, Japan. The authors thank the

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Food Environ Virol (2012) 4:93–101 workers of the DWTPs for their kind cooperation in providing the raw water samples.

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