Cryptosporidium is a protozoan parasite that causes the diarrhoeal disease cryptosporidiosis. It is a key concern for water utilities and health regulators because it is common in source waters, highly transmissible, difficult to inactivate and has limited effective drug treatments. There are 26 species of Cryptosporidium but only two, C. hominis and C. parvum, are of public health concern because they cause >90% of infections. this project evaluated and improved PCR assays for Cryptosporidium detection and developed new novel PCR assays for Cryptosporidium species identification.

Honours Thesis completed by Anna M. Wilson in November 2013.

This project Identified the promising biomarkers shown to be capable for distinguishing between infectious and non-infectious Cryptosporidium oocysts as well as identify species that can/cannot infect humans. A select few candidates were developed into a new PCR based assay capable of identifying a single infective oocyst as well as simultaneously determine the oocyst species/genotype with a rapid turnaround time between 6-8 hrs of oocyst sample receipt (i.e. same day reporting).

Honours Thesis completed by Michael A. Webber in November 2011.

The protozoan parasites, Cryptosporidium and Giardia represent a major public health concern of water utilities in developed nations. In Australia, marsupials, cattle and sheep are the dominant animals inhabiting water catchment areas and contribute a large volume of manure to catchments. Cryptosporidium fayeri, one of the main species identified in marsupials, was identified in a 29-year-old woman in Sydney in 2009 with identical subtypes found in marsupials in the area. There have also been reports of C. parvum and C. hominis (the most common species found in humans), in kangaroos, a wallaby, possums and bandicoots by independent groups, as well as high prevalence’s of zoonotic genotypes of Giardia in marsupials.

This project conducted a comprehensive study of genotypes of Cryptosporidium and Giardia present in marsupials, pre-weaned cattle and sheep and STP sites, over a three-year period to gain a more thorough understanding of the zoonotic risk these parasites pose to humans. In addition to cataloguing the genotypes present using next generation sequencing technologies, researchers also enumerated the numbers of Cryptosporidium and Giardia (oo)cysts present in samples and conducted a survey of farming practices to determine if particular management practices were associated with a higher or lower prevalence of zoonotic genotypes in pre-weaned cattle and sheep.

Cryptosporidium is a waterborne microscopic parasite with different forms at various stages of its lifecycle. One form, the spherical oocyst, is excreted by infected people and transported in rivers and surface waters. In the first part of this research (WaterRA Project 2015) an in vitro cell culture ‘infectivity assay’ test was developed to be able to differentiate between live infectious and dead ‘safe’ oocysts. In the second part of this research the ‘infectivity assay’ was used to examine the frequency and occurrence of infectious cryptosporidia at different stages of wastewater treatment. Samples from five wastewater treatment plants were examined. It was concluded that there is a strong seasonal element to infectious oocyst removal in which winter rainfall and temperatures reduced removal efficacy but that exposure to sunlight is an effective way to inactivate infectious oocysts and make recycled water safe and fit for purpose.

Smaller and regional Wastewater Treatment Plants (WWTPs) have the capacity to recycle wastewater for agricultural use, but the cost of obtaining regulatory approval or ‘accreditation’ is prohibitive. One reason for this is that each WWTP must demonstrate that its processes and operations consistently remove pathogens that cause infectious diseases in humans. Operating conditions include flow rate through the WWTP, and temperature in the activated sludge component of the WWTPs. Although pathogen ‘log removal values (LVR)’ were obtained for a WWTP at 19-20°C in Part I of this project (WQRA project 2001), these values cannot also be attributed to summer temperatures of 26°C. This research determined LRVs for ‘new’ WWTP operating conditions and combined the data with data from Phase I (Project 2001) for analysis. One of the conclusions from Part II was that faster flow rates associated with increased rainfall reduced pathogen LRVs.

Sewage is delivered to wastewater treatment plants (WWTPs) where benign microbial organisms within ‘activated sludge’ vessels contribute to the removal of harmful pathogens from the sewage. The activity and pathogen-removing ability of these helpful organisms is affected by many factors including temperature, numbers of fine particles, pH, ammonia, and the time available to remove the pathogens. Regulatory authorities require at least 90% (one log removal value, LRV) of the pathogens to be removed, but as WWTP operating conditions vary, the LRVs change. This problem led to recognition of the need to develop models capable of predicting relationships between plant operating parameters (such as temperature) and pathogen removal. This research reviewed published reports and datasets, then set up and ran an experimental activated sludge pilot plant to generate data about a range of operating conditions and pathogen removals. These datasets were used to develop models which had only a ‘poor’ predictive value for clostridia but were ‘good’ for giardia and ‘very good to excellent’ for the removal of other pathogens. These models need to be extended with more operating conditions but have the potential to be used to attribute LRVs and for future integration into online real-time monitoring.

E. coli bacteria naturally populate the gastrointestinal tract of humans and animals; they are usually harmless and are commonly excreted. Faeces can also contain harmful microscopic pathogens, and this has led to the assumption that if harmless E. coli are found in water, that the drinking water has been contaminated with faeces that might also have contained pathogens that pose a risk to public health. Using E. coli as an indicator of faecal contamination was recently challenged by the finding that some E. coli strains live, grow and bloom in the environment, and their presence in water might not mean that the water has been contaminated with harmful pathogens. This research examined the environmental conditions associated with E. coli bloom formation in the context of climate-change adaptation and developed multiplex PCR tests which allow the identification of environmental and faecal E. coli. This information was added to a Utility Response Protocol.

‘Microbial source tracking’ (MST) is a technique that aims to identify the animal that excreted faeces and polluted water. There are a number of ways to do this, but the problem is that no one method accurately identifies the origins of faecal pollution in environmental water samples. This research found that faeces could be stored in a freezer or a laboratory -80°C cold-store for up to a month without changing the relative numbers of the different types of bacteria in the samples of faeces. Up to seven faeces samples from different animals were mixed together and examined using 17 techniques to identify the original animals. Three of the most accurate and reliable methods used mitochondrial DNA, the analysis of a bacterial enzyme sequence (beta-glucuronidase), and specific DNA sequences form bacteria known to come from humans, horses and cows. These three types of tests were selected for inclusion in a ‘Toolbox’ from which a combination of methods will allow accurate and reliable management of faecal contaminants in source waters.

Cryptosporidium, a microscopic pathogen, forms infectious oocysts which are removed by specific and targeted water treatments. Oocysts can only be seen by using a microscope but finding an infectious dose of 10 oocysts in a litre of water is like finding a needle in a haystack. Usually a larger volume of water, 1000mL, is filtered to recover all the oocysts into a small volume of 0.1 to 1 mL, because this is small enough to be examined under a microscope. It is scientific practice to add some dead, colour-dyed oocysts to the large volume of water. If all the coloured oocysts are counted on the filter, the recovery is 100%. From this it became clear that there was a problem with oocyst recovery. This research found that different elements reduced recovery from different types of water, for example, iron and silica reduced oocyst recovery from river or groundwaters. The approved method for quantifying the environmental occurrence of oocysts can now be modified to increase recovery, and this change improves analysis and consequent management which further reduces risks to public health.

Cryptosporidium, a microscopic single-cell parasite, forms an “oocyst” with a resistant outer layer analogous to an eggshell. Oocysts survive for a long time in the environment but UV in sunlight, and high temperatures that cause desiccation, kill them. If a mammal drinks water containing live oocysts, they embed in the gut wall and continue their lifecycle until eventually many more oocysts are excreted. There are 26 species of cryptosporidium but only five infect humans, and two; Cryptosporidium hominus and Cryptosporidium parvum, cause approximately 95% of all human infections. C. hominus occurs only in humans, but C. parvum is also found in cattle, sheep, and other animals. The problem is that human-infecting oocysts are excreted by animals in catchments and rain can wash live oocysts into water reservoirs. This research collated peer-reviewed published literature, and information and data collected by the water industry, then characterised the distribution of different Cryptosporidium species in Australian catchments. This led to recognition of a research need to track and predict live and dead oocyst transport during different weather events, and to model and evaluate catchment management initiatives such as excluding cattle from reservoir areas.