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.

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.