The detection of Japanese encephalitis virus in municipal wastewater during an acute disease outbreak.

AIM: To demonstrate the capability of wastewater-based surveillance (WBS) as a tool for detecting potential cases of Japanese Encephalitis Virus (JEV) infection in the community. METHODS AND RESULTS: In this study, we explore the potential of WBS to detect cases of JEV infection by leveraging from an established SARS-CoV-2 wastewater surveillance program. We describe the use of two reverse transcriptase quantitative polymerase chain reaction (RTqPCR) assays targeting JEV to screen archived samples from two wastewater treatment plants (WWTPs). JEV was detected in wastewater samples collected during a timeframe coinciding with a cluster of acute human encephalitis cases, alongside concurrent evidence of JEV detection in mosquito surveillance and the sentinel chicken programs within South Australia's Riverland and Murraylands regions. CONCLUSIONS: Current surveillance measures for JEV encounter multiple constraints, which may miss the early stages of JEV circulation or fail to capture the full extent of transmission. The detection of JEV in wastewater during a disease outbreak highlights the potential WBS has as a complementary layer to existing monitoring efforts forming part of the One Health approach required for optimal disease response and control.

Cryptosporidium Attenuation across the Wastewater Treatment Train: Recycled Water Fit for Purpose.

Compliance with guideline removal targets for Cryptosporidium which do not provide any credit for the inactivation of oocysts through wastewater treatment processes can considerably increase the cost of providing recycled water. Here we present the application of an integrated assay to quantify both oocyst numbers and infectivity levels after various treatment stages at three Victorian and two South Australian (SA) wastewater treatment plants (WWTPs). Oocyst density in the raw sewage was commensurate with community disease burden, with early rounds of sampling capturing a widespread cryptosporidiosis outbreak in Victoria. The level of infectivity of oocysts in sewage was stable throughout the year but was significantly lower at the SA WWTPs. Removals across secondary treatment processes were seasonal, with poorer removals associated with inflow variability; however, no decrease in the oocyst infectivity was identified. For SA WWTPs, those oocysts remaining within the secondary treatment-clarified effluent were proportionally more infectious than those in raw sewage. Lagoon systems demonstrated significant inactivation or removal of oocysts, with attenuation being seasonal. Examination of a UV system emphasized its efficacy as a disinfectant barrier but conversely confirmed the importance of a multibarrier approach with the detection of infectious oocysts postdisinfection. The ability to characterize risk from infectious oocysts revealed that the risk from Cryptosporidium is significantly lower than previously thought and that its inclusion in quantitative risk assessments of reuse systems will more accurately direct the selection of treatment strategies and capital expenditure, influencing the sustainability of such schemes.IMPORTANCE Here we present the application of a recently developed integrated assay not only to quantify the removal of Cryptosporidium oocysts but also to quantify their infectivity across various treatment stages at five wastewater treatment plants (WWTPs), thereby better measuring the "true effect" of the treatment train on oocyst risk reduction. For a number of the WWTPs analyzed in this study the risk, is significantly lower than previously thought. Therefore, the inclusion of oocyst infectivity in guideline values and in quantitative microbial risk assessment (QMRA) has the potential to affect future treatment directions and capital expenditure.

Integrated cryptosporidium assay to determine oocyst density, infectivity, and genotype for risk assessment of source and reuse water.

Cryptosporidium continues to be problematic for the water industry, with risk assessments often indicating that treatment barriers may fail under extreme conditions. However, risk analyses have historically used oocyst densities and not considered either oocyst infectivity or species/genotype, which can result in an overestimation of risk if the oocysts are not human infective. We describe an integrated assay for determining oocyst density, infectivity, and genotype from a single-sample concentrate, an important advance that overcomes the need for processing multiple-grab samples or splitting sample concentrates for separate analyses. The assay incorporates an oocyst recovery control and is compatible with standard primary concentration techniques. Oocysts were purified from primary concentrates using immunomagnetic separation prior to processing by an infectivity assay. Plate-based cell culture was used to detect infectious foci, with a monolayer washing protocol developed to allow recovery and enumeration of oocysts. A simple DNA extraction protocol was developed to allow typing of any wells containing infectious Cryptosporidium. Water samples from a variety of source water and wastewater matrices, including a semirural catchment, wastewater, an aquifer recharge site, and storm water, were analyzed using the assay. Results demonstrate that the assay can reliably determine oocyst densities, infectivity, and genotype from single-grab samples for a variety of water matrices and emphasize the varying nature of Cryptosporidium risk extant throughout source waters and wastewaters. This assay should therefore enable a more comprehensive understanding of Cryptosporidium risk for different water sources, assisting in the selection of appropriate risk mitigation measures.

Cytotoxicity screening for the cyanobacterial toxin cylindrospermopsin.

The cell lines C3A, HepG2, NCI-87, HCT-8, HuTu-80, Caco-2, and Vero were screened for sensitivity to the cyanobacterial toxin cylindrospermopsin (CYN), with the aim of determining the most sensitive cells to be used in cytotoxicity tests. Cell lines were chosen to be representative of the organs targeted by the toxin; liver, kidney, intestine, and were expected to have different metabolic activities and uptake capabilities. Over the range of cell lines tested, IC(50) determinations at 24 h (MTT assay) ranged fourfold, from 1.5 muM for hepatocyte-derived cell lines (C3A IC(50) = 1.5 +/- 0.54; HepG2 IC(50) = 1.5 +/- 0.87) to 6.5 +/- 3.3 micro the colon-derived Caco-2 cell line. The cell-line sensitivity seemed to decrease in cell lines derived from progressively more distal regions of the gastrointestinal tract: gastric > duodenal > ileal > colonic. The greater sensitivity of the hepatic cell lines to CYN was also apparent in 7-d exposure studies, with low toxin concentrations exerting cytotoxic effects that were not seen in other cell lines. Short-term exposure of C3A cells to CYN (1-6 h) was shown to induce cytotoxicity at 24 h despite a washout and recovery incubation, demonstrating the protracted and apparently irreversible nature of CYN's toxic effects.

Bacterial degradation of microcystin toxins in drinking water eliminates their toxicity.

Microcystin-LR and -LA were readily biodegraded by a bacterium, Sphingpoyxis sp. LH21, in a treated reservoir water. Detection of the microcystins was conducted using high-performance liquid chromatography (HPLC), protein phosphatase 2A (PP2A) inhibition assay and a cell-based cytotoxicity assay. The HPLC results correlated well with the two assays. The decrease in cytotoxicity, coupled with the associated decrease in microcystin concentrations, indicated that no cytotoxic by-products were being generated, highlighting the applicability of biodegradation as a feasible treatment option for effective microcystin removal.

Application of the neuroblastoma assay for paralytic shellfish poisons to neurotoxic freshwater cyanobacteria: interlaboratory calibration and comparison with other methods of analysis.

Paralytic shellfish poisons (PSPs) are produced by freshwater cyanobacteria and pose a threat to human and animal drinking-water supplies. The wide range of toxin analogues (and the likelihood that further analogues remain to be discovered) means that chromatographic methods are not always reliable indicators of toxicity. Although the mouse bioassay remains the method of choice in the seafood industry, its use is increasingly being questioned on ethical grounds. The cell-based Neuro-2A neuroblastoma toxicity assay is an alternative bioassay validated for testing shellfish extracts, so it was of interest to determine its applicability with the different suite of toxin analogues produced by cyanobacteria. Cyanobacterial bloom samples from Australia, Brazil, and France were assayed using the neuroblastoma assay, liquid chromatography-tandem mass spectrometry (LC-MS/MS), high-performance liquid chromatography with postcolumn derivatization and fluorescence detection, and the Jellett Rapid Test for PSP. To assess interlaboratory variability, the neuroblastoma assay was set up in laboratories in Paris (France) and Adelaide (Australia). Neuroblastoma and chromatographic methods gave comparable results except in the case of the neurotoxic Brazilian samples: LC-MS/MS did not detect the putative new PSPs contained in these samples. Inter- and intralaboratory variability of the neuroblastoma assay was typical of biological assays but no greater than that found for interassay variability between different chromatographic determinations. The batch of Jellett Rapid Tests for PSP used did not yield quantitative results. Overall, the neuroblastoma assay was useful as a screening assay for determination of toxicity caused by saxitoxin neurotoxins in freshwater cyanobacteria, having the advantage of being sensitive to unidentified toxins that currently cannot be quantified by chromatographic means.

Cell culture-Taqman PCR assay for evaluation of Cryptosporidium parvum disinfection.

Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log(10) units) with LP-UV (20 mJ/cm(2)) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log(10) unit being inactivated. These results demonstrate the inability of MIOX to inactivate Cryptosporidium. The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water.