The Molecular Detection of Cryptosporidium and Giardia in Sika Deer (Cervus Nippon Centralis) in Japan.

Fecal specimens (271 samples) from wild deer, Cervus nippon centralis, were collected from nine different areas in Japan; these samples were subjected to a real-time reverse transcription PCR for Cryptosporidium-and Giardia-specific 18S ribosomal RNA to investigate the prevalence of Cryptosporidium and Giardia infection. The incidence of Cryptosporidium and Giardia in the nine areas ranged from 0% to 20.0% and 0% to 3.4%, respectively. The prevalence of Cryptosporidium among male and female deer was 8.1% and 3.9%, respectively, while that of Giardia was 0.7% and 0.8%. Sequence analysis identified the Cryptosporidium deer genotype, Cryptosporidium bovis, Cryptosporidium ryanae and Cryptosporidium meleagridis from the sequence of Cryptosporidium-specific partial 18S ribosomal RNA and Giardia intestinalis assemblage A from the partial sequence of Giardia-specific 18S rRNA. The variation in regional prevalence indicates that Cryptosporidium infection depends on environmental factors, and that bovine Cryptosporidium was detected more frequently than cervine Cryptosporidium. These data suggest that wild deer might be a healthy carrier of bovine Cryptosporidium.

Community analysis of picocyanobacteria in an oligotrophic lake by cloning 16S rRNA gene and 16S rRNA gene amplicon sequencing.

In this study, the picocyanobacterial species composition of Lake Miyagase was examined by analyzing the 16S rRNA gene in a clone library and by amplicon sequencing using a benchtop next-generation sequencer. Five separate samples were analyzed from different days over a ten-month period. In the picocyanobacterial lineage, 9 and 12 OTUs were identified from a clone library and by amplicon sequencing, respectively. Both analyses suggested that a picocyanobacterium related to Synechococcus sp. MW6B4 was dominant in Lake Miyagase. Our findings suggest that 16S rRNA gene amplicon sequencing enables detailed evaluation of picocyanobacteria composition. One OTU identified was found to be a novel cluster that does not group with any of the known freshwater picocyanobacteria.

Quantitative detection of human enteric adenoviruses in river water by microfluidic digital polymerase chain reaction.

We describe an assay for simple and accurate quantification of human enteric adenoviruses (EAdVs) in water samples using a recently developed quantification method named microfluidic digital polymerase chain reaction (dPCR). The assay is based on automatic distribution of reaction mixture into a large number of nanolitre-volume reaction chambers and absolute copy number quantification from the number of chambers containing amplification products on the basis of Poisson statistics. This assay allows absolute quantification of target genes without the use of standard DNA. Concentrations of EAdVs in Japanese river water samples were successfully quantified by the developed dPCR assay. The EAdVs were detected in seven of the 10 samples (1 L each), and the concentration ranged from 420 to 2,700 copies/L. The quantified values closely resemble those by most probable number (MPN)-PCR and real-time PCR when standard DNA was validated by dPCR whereas they varied substantially when the standard was not validated. Accuracy and sensitivity of the dPCR was higher than those of real-time PCR and MPN-PCR. To our knowledge, this is the first study that has successfully quantified enteric viruses in river water using dPCR. This method will contribute to better understanding of existence of viruses in water.

[Current situation and problems associated with inactivation of microorganisms in water using copper].

OBJECTIVES: The current situation and problems associated with inactivation of microorganisms in water using copper were elucidated. METHODS: A literature review was conducted regarding the history and mechanisms of inactivation technology using copper, the variety of microorganisms shown to be inactivated by these methods in previous experiments, and the efficacy of such technologies for the inactivation of microorganisms in water. RESULTS: The use of copper for inactivation of microorganisms has a long history. Although the use of copper was discontinued temporarily owing to the advent of antibiotics in the 1930s, the occurrence of antibiotic-resistant bacteria has resulted in the need for different approaches to control pathogenic microorganisms. One such alternative is the use of copper. Although the mechanisms underlying the efficacy of copper inactivation technology have not yet been elucidated in detail, it has been suggested that pathogenic bacteria are inactivated due to the toxicity of copper ions and strong oxidation effects of reactive oxygen species. Copper inactivation technology is effective against many pathogenic microorganisms that pose a risk to public health, such as Legionella pneumophila, Salmonella enterica, and Mycobacterium tuberculosis. In recent years, copper inactivation technology has been used in various water-related devices, especially water supply pipes in buildings. Previous studies have demonstrated that microorganisms can be sufficiently inactivated by copper even at concentrations below that specified in the Water Quality Standard for Drinking Water. However, some previous studies have indicated that the inactivation effect of copper is short-lived. Therefore, the development of techniques to maintain a long-term inactivation effect is a key concern. In addition, it has been reported that the use of copper pipes triggers chlorine decay and results in the formation of chlorine disinfection byproducts. Hence, further studies should aim at assessing the risks and benefits associated with the use of copper. CONCLUSION: Although the practical issues regarding copper inactivation technology are persistent, this method has been demonstrated to be efficacious. Therefore, this technology could be expected to be used in many devices such as water supply systems in hospitals in the near future.

Occurrence of pepper mild mottle virus in drinking water sources in Japan.

Pepper mild mottle virus (PMMoV) is a plant virus that has been recently proposed as a potential indicator of human fecal contamination of environmental waters; however, information on its geographical occurrence in surface water is still limited. We aimed to determine the seasonal and geographic occurrence of PMMoV in drinking water sources all over Japan. Between July 2008 and February 2011, 184 source water samples were collected from 30 drinking water treatment plants (DWTPs); viruses from 1 to 2 liters of each sample were concentrated by using an electronegative membrane, followed by RNA extraction and reverse transcription. Using quantitative PCR, PMMoV was detected in 140 (76%) samples, with a concentration ranging from 2.03×10(3) to 2.90×10(6) copies/liter. At least one of the samples from 27 DWTPs (n=4 or 8) was positive for PMMoV; samples from 10 of these DWTPs were always contaminated. There was a significant difference in the occurrence of PMMoV among geographical regions but not a seasonal difference. PMMoV was frequently detected in samples that were negative for human enteric virus or Escherichia coli. A phylogenetic analysis based on the partial nucleotide sequences of the PMMoV coat protein gene in 12 water samples from 9 DWTPs indicated that there are genetically diverse PMMoV strains present in drinking water sources in Japan. To our knowledge, this is the first study to demonstrate the occurrence of PMMoV in environmental waters across wide geographical regions.

Serum C1q- binding adiponectin in maintenance hemodialysis patients.

BACKGROUND: Patients on maintenance hemodialysis (HD) have much higher levels of adiponectin (Total-APN). Adiponectin and C1q form a protein complex in human blood, and serum C1q-binding adiponectin (C1q-APN) can be measured. We recently reported that C1q-APN/Total-APN ratio rather than Total-APN correlated with atherosclerosis in diabetics. However, the characteristics of C1q-APN in HD patients remain unclear. The preset study investigated the characteristics of the adiponectin parameters including C1q-APN and also to clarify the relationship between various serum adiponectin parameters and atherosclerotic cardiovascular diseases (ACVD) in HD patients. METHODS: The single cross-sectional study subjects were 117 Japanese patients (males/females = 61/56) on regular HD. Blood Total-APN, high molecular weight-adiponectin (HMW-APN), C1q-APN and C1q concentrations were measured by enzyme-linked immunosorbent assays. ACVD were defined as stroke, coronary and peripheral artery diseases, thoracic and abdominal aneurysms. RESULTS: Stepwise regression analysis identified high-density lipoprotein-cholesterol (HDL-C) as the only significant and independent determinant of C1q-APN in males, and duration of HD as the only significant and independent determinant of C1q-APN in females. Stepwise regression analysis identified uric acid, low-density lipoprotein-cholesterol and triglyceride as significant and independent determinants of C1q-APN/Total-APN ratio in males, and leukocyte count and HDL-C as significant and independent determinants of C1q-APN/Total-APN ratio in females. Multiple logistic regression analysis identified inorganic phosphorus and C1q-APN or C1q-APN/C1q ratio as significant determinants of ACVD. CONCLUSIONS: Low serum C1q-APN and C1q-APN/C1q ratio, but not C1q-APN/Total-APN ratio, correlated with ACVD in HD patients. TRIAL REGISTRATION: ClinicalTrials.gov: UMIN http://000004318.

Removal of radioactive iodine and cesium in water purification processes after an explosion at a nuclear power plant due to the Great East Japan Earthquake.

The presence of radionuclides at five water purification plants was investigated after an explosion at a nuclear power plant hit by the Great East Japan Earthquake on 11 March 2011. Radioactive iodine (¹³¹I) and cesium (¹³4Cs and ¹³7Cs) were detected in raw water in Fukushima and neighboring prefectures. ¹³¹I was not removed by coagulation-flocculation-sedimentation. ¹³¹I was removed by granular activated carbon (GAC) and powdered activated carbon (PAC) at a level of about 30%-40%, although ¹³¹I was not removed in some cases. This was also confirmed by laboratory-scale experiments using PAC. The removal percentages of ¹³¹I in river and pond waters by 25 mg dry/L of PAC increased from 36% to 59% and from 41% to 48%, respectively, with chlorine dosing before PAC. ¹³4Cs and ¹³7Cs were effectively removed by coagulation at both a water purification plant and in laboratory-scale experiments when turbidity was relatively high. In contrast, ¹³4Cs and ¹³7Cs in pond water with low turbidity were not removed by coagulation. This was because ¹³4Cs and ¹³7Cs in river water were present mainly in particulate form, while in pond water they were present mainly as cesium ions (¹³4Cs+ and ¹³7Cs+). However, the removal of ¹³4Cs and ¹³7Cs in pond water by coagulation increased markedly when ¹³4Cs and ¹³7Cs were mixed with sediment 24 h before coagulation.

One-year weekly survey of noroviruses and enteric adenoviruses in the Tone River water in Tokyo metropolitan area, Japan.

To investigate the actual fluctuations in the concentrations of noroviruses (NoVs) GI and GII, and enteric adenoviruses (EAdVs) in river water and its relationship with the number of acute infectious gastroenteritis patients, one-year weekly quantitative monitoring of NoVs GI and GII and EAdVs was performed in the Tone River in Japan where the surface water is utilized for the main production of drinking water for the Tokyo Metropolitan Area from October 2009 to September 2010. Noroviruses GI and GII and EAdVs were detected in 28 (54%), 33 (63%), and 23 (44%) of the 52 samples (1 L each), respectively. The concentrations of NoVs GI and GII and EAdVs fluctuated strongly and were more abundant in winter and early spring. The concentration of NoVs GI was transiently greater than 10,000 copies/L. The number of acute infectious gastroenteritis patients in the upper river basin was highly correlated with all the viral concentrations, while general microbial indicator data such as turbidity and heterotrophic plate count were independent of viral concentration as suggested in previous studies. To the best of our knowledge, this is the first study that clearly shows the strong correlation of the number of gastroenteritis with virus contamination in lower river basin.

Rapid start-up of a nitrifying reactor using aerobic granular sludge as seed sludge.

In this study, the effectiveness of aerobic granular sludge as seed sludge for rapid start-up of nitrifying processes was investigated using a laboratory-scale continuous stirred-tank reactor (CSTR) fed with completely inorganic wastewater which contained a high concentration of ammonia. Even when a large amount of granular biomass was inoculated in the reactor, and the characteristics of influent wastewater were abruptly changed, excess biomass washout was not observed, and biomass concentration was kept high at the start-up period due to high settling ability of the aerobic granular sludge. As a result, an ammonia removal rate immediately increased and reached more than 1.0 kg N/m(3)/d within 20 days and up to 1.8 kg N/m(3)/d on day 39. Subsequently, high rate nitritation was stably attained during 100 days. However, nitrite accumulation had been observed for 140 days before attaining complete nitrification to nitrate. Fluorescence in situ hybridization analysis revealed the increase in amount of ammonia-oxidizing bacteria which existed in the outer edge of the granular sludge during the start-up period. This microbial ecological change would make it possible to attain high rate ammonia removal.

Occurrence of viruses and protozoa in drinking water sources of Japan and their relationship to indicator microorganisms.

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 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.

Quantitative detection of Cryptosporidium oocyst in water source based on 18S rRNA by alternately binding probe competitive reverse transcription polymerase chain reaction (ABC-RT-PCR).

We describe an assay for simple and cost-effective quantification of Cryptosporidium oocysts in water samples using a recently developed quantification method named alternately binding probe competitive PCR (ABC-PCR). The assay is based on the detection of 18S rRNA specific for Cryptosporidium oocysts. The standard curve of the ABC-PCR assay had a good fitting to a rectangular hyperbola with a correlation coefficient (R) of 0.9997. Concentrations of Cryptosporidium oocysts in real river water samples were successfully quantified by the ABC-reverse transcription (RT)-PCR assay. The quantified values by the ABC-RT-PCR assay very closely resemble those by the real-time RT-PCR assay. In addition, the quantified concentration in most water samples by the ABC-RT-PCR assay was comparable to that by conventional microscopic observation. Thus, Cryptosporidium oocysts in water samples can be accurately and specifically determined by the ABC-RT-PCR assay. As the only equipment that is needed for this end-point fluorescence assay is a simple fluorometer and a relatively inexpensive thermal cycler, this method can markedly reduce time and cost to quantify Cryptosporidium oocysts and other health-related water microorganisms.

Foaming control by automatic carbon source adjustment using an ORP profile in sequencing batch reactors for enhanced nitrogen removal in swine wastewater treatment.

Incomplete denitrification and ammonia accumulation were found to cause proliferation of filamentous microorganisms in sequencing batch reactors (SBRs) for swine wastewater treatment. Foaming was observed in response to the accumulation of 115.2 and 12.2 mg/L of nitrate and ammonia, respectively. The mixed liquor suspended solids (MLSS) level in SBRs was decreased to 2,000 mg/L and the suspended solids in the effluent reached 200 mg/L when foaming appeared. However, the use of swine waste as an external carbon source for enhanced biological nitrogen removal was found to effectively control the foaming caused by filamentous microorganisms. Therefore, an optimum strategy for the addition of swine waste was designed using integrated real-time control to provide pulse input control of slurry based on the "nitrate knee" in the oxidation-reduction potential profile. In this case, the MLSS concentration was maintained at an average value of approximately 7,550 mg/L, while the SS in the effluent was less than 30 mg/L.

Anaerobic/oxic/anoxic granular sludge process as an effective nutrient removal process utilizing denitrifying polyphosphate-accumulating organisms.

In a biological nutrient removal (BNR) process, the utilization of denitrifying polyphosphate-accumulating organisms (DNPAOs) has many advantages such as effective use of organic carbon substrates and low sludge production. As a suitable process for the utilization of DNPAOs in BNR, an anaerobic/oxic/anoxic granular sludge (AOAGS) process was proposed in this study. In spite of performing aeration for nitrifying bacteria, the AOAGS process can create anaerobic/anoxic conditions suitable for the cultivation of DNPAOs because anoxic zones exist inside the granular sludge in the oxic phase. Thus, DNPAOs can coexist with nitrifying bacteria in a single reactor. In addition, the usability of DNPAOs in the reactor can be improved by adding the anoxic phase after the oxic phase. These characteristics enable the AOAGS process to attain effective removal of both nitrogen and phosphorus. When acetate-based synthetic wastewater (COD: 600 mg/L, NH4-N: 60 mg/L, PO(4)-P: 10 mg/L) was supplied to a laboratory-scale sequencing batch reactor under the operation of anaerobic/oxic/anoxic cycles, granular sludge with a diameter of 500 microm was successfully formed within 1 month. Although the removal of both nitrogen and phosphorus was almost complete at the end of the oxic phase, a short anoxic period subsequent to the oxic phase was necessary for further removal of nitrogen and phosphorus. As a result, effluent concentrations of NH(4)-N, NO(x)-N and PO(4)-P were always lower than 1 mg/L. It was found that penetration depth of oxygen inside the granular sludge was approximately 100 microm by microsensor measurements. In addition, from the microbiological analysis by fluorescence in situ hybridization, existence depth of polyphosphate-accumulating organisms was further than the maximum oxygen penetration depth. The water quality data, oxygen profiles and microbial community structure demonstrated that DNPAOs inside the granular sludge may be responsible for denitrification in the oxic phase, which enables effective nutrient removal in the AOAGS process.

Integrated real-time control strategy for nitrogen removal in swine wastewater treatment using sequencing batch reactors.

A new integrated real-time control system was designed and operated with fluctuating influent loads for swine wastewater treatment. The system was operated with automatic addition control of an external carbon source, using real-time control technology, which utilized the oxidation-reduction potential (ORP) and the pH as parameters to control the anoxic phase and oxic phase, respectively. The fluctuations in swine wastewater concentration are extreme; an influent with a low C/N ratio is deficient in organic carbon, and a low carbon source level can limit the overall biological denitrification process. Consequently, a sufficient organic source must be provided for proper denitrification. The feasibility of using swine waste as an external carbon source for enhanced biological nitrogen removal was investigated. The real-time control made it possible to optimize the quantity of swine waste added as the load fluctuated from cycle to cycle. The average removal efficiencies achieved for TOC and nitrogen were over 94% and 96%, respectively, using the integrated real-time control strategy.

Effectiveness of oxidation-reduction potential and pH as monitoring and control parameters for nitrogen removal in swine wastewater treatment by sequencing batch reactors.

Two bench-scale sequencing batch reactors (SBRs) were operated in a fixed hydraulic retention time study to investigate the effectiveness of oxidation-reduction potential (ORP), pH and dissolved oxygen as parameters for indicating denitrification followed by nitrification in SBRs for swine wastewater treatment. The ORP and pH profiles were monitored and evaluated under different denitrification and nitrification conditions with and without a supplemental carbon source. With a low C/N ratio, and using a suitable C/N ratio adjustment control, ORP and pH could be used as monitoring and control parameters in both the anoxic and oxic phases for practical swine wastewater treatment. High-level accumulation of nitrate was observed without any C/N ratio adjustment. In this case, ORP and pH were not useful for monitoring denitrification followed by nitrification in SBRs. According to our research, with regard to N removal, it would be better to use pH as a parameter during the oxic phase and ORP as a parameter during the anoxic phase. Using a suitable adjustment of a ON ratio in the influent by adding swine slurry, a high total nitrogen removal efficiency of up to 95.5% was reached. It was found that, in this case, the use of ORP and pH as parameters for real-time control processes was possible in swine wastewater treatment.