The efficacy of current treatment processes to remove, inactivate, or reduce environmental bloom-forming Escherichia coli.

Escherichia coli is excreted in high numbers from the intestinal tract of humans, other mammals, and birds. Traditionally, it had been thought that E. coli could grow only within human or animal hosts and would perish in the environment. Therefore, the presence of E. coli in water has become universally accepted as a key water quality indicator of fecal pollution. However, recent research challenges the assumption that the presence of E. coli in water is always an indicator of fecal contamination, with some types of E. coli having evolved to survive and grow in aquatic environments. These strains can form blooms in water storages, resulting in high E. coli counts even without fecal contamination. Although these bloom-forming strains lack virulence genes and pose little threat to public health, their presence in treated water triggers the same response as fecal-derived E. coli. Yet, little is known about the effectiveness of treatment processes in removing or inactivating them. This study evaluated the effectiveness of current treatment processes to remove bloom-forming strains, in comparison to fecal-derived strains, with conventional coagulation-flocculation-sedimentation and filtration investigated. Second, the effectiveness of current disinfection processes-chlorination, chloramination, and ultraviolet (UV) light to disinfect bloom-forming strains in comparison to fecal-derived strains-was assessed. These experiments showed that the responses of bloom isolates were not significantly different from those of fecal E. coli strains. Therefore, commonly used water treatment and disinfection processes are effective to remove bloom-forming E. coli strains from water.IMPORTANCEThe presence of Escherichia coli in water has long been used globally as a key indicator of fecal pollution and for quantifying water safety. Traditionally, it was believed that E. coli could only thrive within hosts and would perish outside, making its presence in water indicative of fecal contamination. However, recent research has unveiled strains of E. coli capable of surviving and proliferating in aquatic environments, forming blooms even in the absence of fecal contamination. While these bloom-forming strains lack the genes to be pathogenic, their detection in source or drinking water triggers the same response as fecal-derived E. coli. Yet, little is known about the efficacy of treatment processes in removing them. This study evaluated the effectiveness of conventional treatment and disinfection processes in removing bloom-forming strains compared to fecal-derived strains. Results indicate that these commonly used processes are equally effective against both types of E. coli, reassuring that bloom-forming E. coli strains can be eliminated from water.

Comparison of kits for SARS-CoV-2 extraction in liquid and passive samples.

Effective extraction and detection of viral nucleic acids from sewage are fundamental components of a successful SARS-CoV-2 sewage surveillance programme. As there is no standard method employed in sewage surveillance, understanding the performance of different extraction kits in the recovery of SARS-CoV-2 and the impact that PCR inhibitors have on quantification is essential to minimize data discrepancies caused by sample extraction. Three commercial nucleic acid extraction kits: the RNeasy PowerSoil Total RNA Kit (PS), the RNeasy PowerMicrobiome Kit (PMB), and the MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit (MM), with minor modifications, were evaluated. Their efficacy in recovering viral ribonucleic acid and removal of PCR inhibitors was assessed using two South Australian wastewater matrices-one from a major metropolitan site and one from a regional centre. Both had SARS-CoV-2 present due to active COVID-19 cases in these communities. Overall, the MM kit had a higher recovery of SARS-CoV-2 from the samples tested, followed by PMB and PS. The PMB kit performance was strongly influenced by the sample matrix when compared to the MM kit. It is recommended to assess the performance of extraction kits using different local wastewater matrices to ensure the accuracy and reliability of monitoring results to avoid false reporting.

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.

Comparison of in vitro growth characteristics of Cryptosporidium hominis (IdA15G1) and Cryptosporidium parvum (Iowa-IIaA17G2R1 and IIaA18G3R1).

Cryptosporidium is a major cause of diarrhoeal disease and mortality in young children in resource-poor countries, for which no vaccines or adequate therapeutic options are available. Infection in humans is primarily caused by two species: C. hominis and C. parvum. Despite C. hominis being the dominant species infecting humans in most countries, very little is known about its growth characteristics and life cycle in vitro, given that the majority of our knowledge of the in vitro development of Cryptosporidium has been based on C. parvum. In the present study, the growth and development of two C. parvum isolates (subtypes Iowa-IIaA17G2R1 and IIaA18G3R1) and one C. hominis isolate (subtype IdA15G1) in HCT-8 cells were examined and compared at 24 h and 48 h using morphological data acquired with scanning electron microscopy. Our data indicated no significant differences in the proportion of meronts or merozoites between species or subtypes at either time-point. Sexual development was observed at the 48-h time-point across both species through observations of both microgamonts and macrogamonts, with a higher frequency of macrogamont observations in C. hominis (IdA15G1) cultures at 48-h post-infection compared to both C. parvum subtypes. This corresponded to differences in the proportion of trophozoites observed at the same time point. No differences in proportion of microgamonts were observed between the three subtypes, which were rarely observed across all cultures. In summary, our data indicate that asexual development of C. hominis is similar to that of C. parvum, while sexual development is accelerated in C. hominis. This study provides new insights into differences in the in vitro growth characteristics of C. hominis when compared to C. parvum, which will facilitate our understanding of the sexual development of both species.

Organoids and Bioengineered Intestinal Models: Potential Solutions to the Cryptosporidium Culturing Dilemma.

Cryptosporidium is a major cause of severe diarrhea-related disease in children in developing countries, but currently no vaccine or effective treatment exists for those who are most at risk of serious illness. This is partly due to the lack of in vitro culturing methods that are able to support the entire Cryptosporidium life cycle, which has led to research in Cryptosporidium biology lagging behind other protozoan parasites. In vivo models such as gnotobiotic piglets are complex, and standard in vitro culturing methods in transformed cell lines, such as HCT-8 cells, have not been able to fully support fertilization occurring in vitro. Additionally, the Cryptosporidium life cycle has also been reported to occur in the absence of host cells. Recently developed bioengineered intestinal models, however, have shown more promising results and are able to reproduce a whole cycle of infectivity in one model system. This review evaluates the recent advances in Cryptosporidium culturing techniques and proposes future directions for research that may build upon these successes.

Understanding human infectious Cryptosporidium risk in drinking water supply catchments.

Treating drinking water appropriately depends, in part, on the robustness of source water quality risk assessments, however quantifying the proportion of infectious, human pathogenic Cryptosporidium oocysts remains a significant challenge. We analysed 962 source water samples across nine locations to profile the occurrence, rate and timing of infectious, human pathogenic Cryptosporidium in surface waters entering drinking water reservoirs during rainfall-runoff conditions. At the catchment level, average infectivity over the four-year study period reached 18%; however, most locations averaged <5%. The maximum recorded infectivity fraction within a single rainfall runoff event was 65.4%, and was dominated by C. parvum. Twenty-two Cryptosporidium species and genotypes were identified using PCR-based molecular techniques; the most common being C. parvum, detected in 23% of water samples. Associations between landuse and livestock stocking characteristics with Cryptosporidium were determined using a linear mixed-effects model. The concentration of pathogens in water were significantly influenced by flow and dominance of land-use by commercial grazing properties (as opposed to lifestyle properties) in the catchment (p < 0.01). Inclusion of measured infectivity and human pathogenicity data into a quantitative microbial risk assessment (QMRA) could reduce the source water treatment requirements by up to 2.67 log removal values, depending on the catchment, and demonstrated the potential benefit of collating such data for QMRAs.

Science: How the Status Quo Harms its Cultural Authority.

Three distinct explanatory models are described which underpin the relationship between the cultural authority of science and public trust. This essay describes how current discourses framed around how the enterprise of science is undertaken; damage these models, diminishing knowledge-attitudes, alienating the public while reducing the cultural meaning of science.

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.

Zoonotic Cryptosporidium Species in Animals Inhabiting Sydney Water Catchments.

Cryptosporidium is one of the most common zoonotic waterborne parasitic diseases worldwide and represents a major public health concern of water utilities in developed nations. As animals in catchments can shed human-infectious Cryptosporidium oocysts, determining the potential role of animals in dissemination of zoonotic Cryptosporidium to drinking water sources is crucial. In the present study, a total of 952 animal faecal samples from four dominant species (kangaroos, rabbits, cattle and sheep) inhabiting Sydney's drinking water catchments were screened for the presence of Cryptosporidium using a quantitative PCR (qPCR) and positives sequenced at multiple loci. Cryptosporidium species were detected in 3.6% (21/576) of kangaroos, 7.0% (10/142) of cattle, 2.3% (3/128) of sheep and 13.2% (14/106) of rabbit samples screened. Sequence analysis of a region of the 18S rRNA locus identified C. macropodum and C. hominis in 4 and 17 isolates from kangaroos respectively, C. hominis and C. parvum in 6 and 4 isolates respectively each from cattle, C. ubiquitum in 3 isolates from sheep and C. cuniculus in 14 isolates from rabbits. All the Cryptosporidium species identified were zoonotic species with the exception of C. macropodum. Subtyping using the 5' half of gp60 identified C. hominis IbA10G2 (n = 12) and IdA15G1 (n = 2) in kangaroo faecal samples; C. hominis IbA10G2 (n = 4) and C. parvum IIaA18G3R1 (n = 4) in cattle faecal samples, C. ubiquitum subtype XIIa (n = 1) in sheep and C. cuniculus VbA23 (n = 9) in rabbits. Additional analysis of a subset of samples using primers targeting conserved regions of the MIC1 gene and the 3' end of gp60 suggests that the C. hominis detected in these animals represent substantial variants that failed to amplify as expected. The significance of this finding requires further investigation but might be reflective of the ability of this C. hominis variant to infect animals. The finding of zoonotic Cryptosporidium species in these animals may have important implications for the management of drinking water catchments to minimize risk to public health.

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.

Investigating source water Cryptosporidium concentration, species and infectivity rates during rainfall-runoff in a multi-use catchment.

Protozoan pathogens present a significant human health concern, and prevention of contamination into potable networks remains a key focus for drinking water providers. Here, we monitored the change in Cryptosporidium concentration in source water during high flow events in a multi-use catchment. Furthermore, we investigated the diversity of Cryptosporidium species/genotypes present in the source water, and delivered an oocyst infectivity fraction. There was a positive and significant correlation between Cryptosporidium concentration and flow (ρ = 0.756) and turbidity (ρ = 0.631) for all rainfall-runoff events, despite variable source water pathogen concentrations. Cell culture assays measured oocyst infectivity and suggested an overall source water infectious fraction of 3.1%. No infectious Cryptosporidium parvum or Cryptosporidium hominis were detected, although molecular testing detected C. parvum in 7% of the samples analysed using PCR-based molecular techniques. Twelve Cryptosporidium species/genotypes were identified using molecular techniques, and were reflective of the host animals typically found in remnant vegetation and agricultural areas. The inclusion of molecular approaches to identify Cryptosporidium species and genotypes highlighted the diversity of pathogens in water, which originated from various sources across the catchment. We suggest this mixing of runoff water from a range of landuses containing diverse Cryptosporidium hosts is a key explanation for the often-cited difficulty forming strong pathogen-indicator relationships.

Solar radiation induces non-nuclear perturbations and a false start to regulated exocytosis in Cryptosporidium parvum.

Stratospheric ozone depletion, climate warming and acidification of aquatic ecosystems have resulted in elevated levels of solar radiation reaching many aquatic environments with an increased deleterious impact on a wide range of living organisms. While detrimental effects on living organisms are thought to occur primarily through DNA damage, solar UV can also damage cellular proteins, lipids and signalling pathways. Cryptosporidium, a member of the eukaryotic phylum Apicomplexa, contain numerous vesicular secretory organelles and their discharge via regulated exocytosis is essential for the successful establishment of infection. Using flow cytometric techniques we demonstrate that solar UV rapidly induces sporozoite exocytosis resulting in a significant reduction in the ability of sporozoites to attach and invade host cells. We found that solar UV induced sporozoite membrane depolarization, resulting in reduced cellular ATP and increased cytosolic calcium. These changes were accompanied by a reduction in the internal granularity of sporozoites, indicative of apical organelle discharge, which was confirmed by analysis of sporozoites with an exocytosis-sensitive dye. The precise timing of apical organelle discharge in the presence of a compatible host cell is critical for sporozoite attachment and invasion. Our results demonstrate for the first time how solar UV radiation can interfere with exocytosis, a fundamental cellular process in all eukaryotic cells. We contend that not only may the forecast increases in solar radiation in both aquatic and terrestrial environments significantly affect members of the Apicomplexa, solar UV-induced membrane depolarizations resulting in cytosolic calcium perturbation may affect a wider range of eukaryotic organisms through antagonistic effects on a myriad of calcium dependant cellular functions.

Environmental temperature controls Cryptosporidium oocyst metabolic rate and associated retention of infectivity.

Cryptosporidium is a significant cause of water-borne enteric disease throughout the world and represents a challenge to the water industry and a threat to public health. In this study we report the use of a cell culture-TaqMan PCR assay to measure oocyst inactivation rates in reagent-grade and environmental waters over a range of temperatures. While oocysts incubated at 4 degrees C and 15 degrees C remained infective over the 12-week holding period, we observed a 4 log(10) reduction in infectivity for both 20 and 25 degrees C incubation treatments at 12 and 8 weeks, respectively, for all water types examined, a faster rate of inactivation for oocysts than previously reported. This temperature-dependent inactivation was further investigated using a simple and rapid ATP assay described herein. Time course experiments performed in reagent-grade water at incubation temperatures of 4, 15, 20, 25, 30, and 37 degrees C identified a close relationship between oocyst infectivity and oocyst ATP content, demonstrating that temperature inactivation at higher temperatures is a function of increased oocyst metabolic activity. While water quality did not affect oocyst inactivation, biological antagonism appears to be a key factor affecting oocyst removal from environmental waters. Both the cell culture-TaqMan PCR assay and the ATP assay provide a sensitive and quantitative method for the determination of environmental oocyst inactivation, providing an alternative to the more costly and time-consuming mouse infection assay. The findings presented here relating temperature to oocyst inactivation provide valuable information for determining the relative risks associated with Cryptosporidium oocysts in water.

The CesA gene family of barley. Quantitative analysis of transcripts reveals two groups of co-expressed genes.

Sequence data from cDNA and genomic clones, coupled with analyses of expressed sequence tag databases, indicate that the CesA (cellulose synthase) gene family from barley (Hordeum vulgare) has at least eight members, which are distributed across the genome. Quantitative polymerase chain reaction has been used to determine the relative abundance of mRNA transcripts for individual HvCesA genes in vegetative and floral tissues, at different stages of development. To ensure accurate expression profiling, geometric averaging of multiple internal control gene transcripts has been applied for the normalization of transcript abundance. Total HvCesA mRNA levels are highest in coleoptiles, roots, and stems and much lower in floral tissues, early developing grain, and in the elongation zone of leaves. In most tissues, HvCesA1, HvCesA2, and HvCesA6 predominate, and their relative abundance is very similar; these genes appear to be coordinately transcribed. A second group, comprising HvCesA4, HvCesA7, and HvCesA8, also appears to be coordinately transcribed, most obviously in maturing stem and root tissues. The HvCesA3 expression pattern does not fall into either of these two groups, and HvCesA5 transcript levels are extremely low in all tissues. Thus, the HvCesA genes fall into two general groups of three genes with respect to mRNA abundance, and the co-expression of the groups identifies their products as candidates for the rosettes that are involved in cellulose biosynthesis at the plasma membrane. Phylogenetic analysis allows the two groups of genes to be linked with orthologous Arabidopsis CesA genes that have been implicated in primary and secondary wall synthesis.