Una Ryan

Cryptosporidium species are an important cause of diarrhoeal disease worldwide. Many countries reported declines in cryptosporidiosis incidence during COVID-19 restrictions, followed by marked increases post-COVID. A similar pattern has been observed in Australia, with record-high case numbers reported across multiple states in 2024–2025, including Western Australia (WA), where an outbreak occurred in 2025. Between 1st January – 30th June 2025, WA reported a statewide total of 1110 human cryptosporidiosis cases, which is substantially higher than the previous years. In the present study, a subset of WA cases from 2025 was investigated using contact tracing and molecular typing and compared with subtypes circulating in 2023 and 2024. Sequence analysis of the 18S rRNA and gp60 loci identified three Cryptosporidium species: C. hominis (91.5 %, 108/118), C. parvum (6.8 %, 8/118), and C. meleagridis (1.7 %, 2/118), comprising 13 distinct subtypes, over the three years. Notably, a previously rare C. hominis subtype, IeA11G3T3, emerged in 2023 and by 2025 had become the dominant subtype (92.1 %, 70/76) of samples typed. Multi-locus sequence typing (MLST) of a representative subset of IeA11G3T3 isolates (n = 13) showed that all sequences were identical, except for one isolate from 2024, which contained three insertions: an 18-bp repeat in the cp-47 gene and both a 12-bp and a 6-bp repeat in the dz-hrgp gene. The C. hominis IeA11G3T3 subtype has also recently emerged as a dominant subtype in several other countries, although the factors driving its emergence remain unclear.

Cattle infected with Cryptosporidium can shed large quantities of the environmentally resistant oocysts, which can cause significant diarrhoeal disease, particularly in neonatal calves and in susceptible human populations worldwide. More than ten species of Cryptosporidium have been reported in cattle; however, C. parvum dominates in young calves in many countries, with C. ryanae, C. bovis and C. andersoni prevalent in older animals. Cryptosporidium hominis and C. parvum are the main species infecting humans. In most countries, zoonotic cryptosporidiosis is primarily caused by C. parvum IIa subtypes, which also dominates in calves, but in China, C. parvum infections in cattle are exclusively caused by IId subtypes. Outbreak investigations and molecular epidemiological studies support calves as a major source of zoonotic cryptosporidiosis. The zoonotic significance of increasing reports of C. hominis in cattle requires further investigation. Epidemiological investigations designed to better understand the sources and transmission dynamics using improved typing tools are required before better control strategies can be implemented.

The major drinking water reservoirs and catchments supplying many of Australia's capital cities have been protected from significant levels of public access since their construction (up to 140 years ago). In addition to the primary intended initial benefit, (protecting drinking water quality to prevent typhoid and similar disease outbreaks), additional benefits included reduced flooding, improved and more stable yield, reduced water treatment costs, and protection of native ecosystems. In relation to the latter benefit, over that period, much of the Australian landscape has been modified for various forms of development and recreational activities, leaving these water catchments as some of the last broad areas of remnant habitat for vulnerable and endangered ecosystems and species. Despite these widely appreciated and well-understood benefits, there has been continuous pressure from a diverse range of interest groups to open these areas. As government-owned organisations, the Australian water utilities consider all such requests from the community. Among the interest groups that make representations to access drinking water sources are a wide range of recreators. Pressure from such groups is increasing as populations grow. To help inform decisions on how to respond to such representations, this study predicted gastrointestinal disease burdens from recreation on a currently protected drinking water supply reservoir in Australia. This study considered a range of scenarios, described in terms understandable to the community, and predicted health implications using screening-level quantitative microbial risk assessment (QMRA). The assessment was limited to microbial risks to drinking water quality - risks from chemical or physical hazards were not considered, nor was ecosystem protection. The QMRA predicted that six reasonably foreseeable scenarios could result in microbial risks that exceeded the health-based target benchmark given in the Australian Drinking Water Guidelines (ADWG). Therefore, additional water treatment would be required to reduce those risks to acceptable levels. However, even with the introduction of additional treatment, permitting increases in the levels of recreational activity in the source water was found to be inconsistent with many of the Guiding Principles of the ADWG and with a landmark state Supreme Court planning decision that had interpreted how those principles should be applied in drinking water catchments. Therefore, the results did not support permitting recreational access to the reservoir, and the importance of source protection was reinforced.

Giardia is the most common protozoan cause of diarrhoeal illness in humans worldwide. Despite this, our understanding of the zoonotic transmission of Giardia, and in particular the role of cattle as a zoonotic reservoir, is not well understood, due to the limitations of current typing systems and a recent taxonomic revision of the genus. Newly improved multilocus sequencing typing tools are not yet widely used and are not applicable to all species. However, data generated to date suggest that zoonotic transmission of Giardia of bovine origin is limited. Carefully designed epidemiological investigations using improved typing tools are essential to understand the extent of zoonotic transmission from cattle. Improved on-farm biosecurity measures are also needed to control the transmission of zoonotic Giardia in cattle.

The enteric parasite Cryptosporidium remains a treatment challenge for drinking water utilities globally due to its resistance to chlorine disinfection. However, the lack of an in vitro culture system for Cryptosporidium that is both cost-effective and reliable remains a key bottleneck in Cryptosporidium research. Here we report that the microfluidic culture of HCT-8 cells under fluid shear stress enables the extended development of Cryptosporidium parvum. Specifically, the growth of C. parvum in a user-friendly pumpless microfluidic device was assessed using immunofluorescence assays, scanning electron microscopy and quantitative PCR, which revealed that development continued for ten days in total. Oocysts produced within the microfluidic device were infective to fresh HCT-8 monolayers, however these oocysts were only present at low levels. We anticipate that such microfluidic approaches will facilitate a wide range of in vitro studies on Cryptosporidium and may have the potential to be further developed as a routine infectivity assessment tool for the water industry.

Marsupials, inhabiting diverse ecosystems, including urban and peri-urban regions in Australasia and the Americas, intersect with human activities, leading to zoonotic spill-over and anthroponotic spill-back of pathogens, including Cryptosporidium and Giardia. This review assesses the current knowledge on the diversity of Cryptosporidium and Giardia species in marsupials, focusing on the potential zoonotic risks. Cryptosporidium fayeri and C. macropodum are the dominant species in marsupials, while in possums, the host-specific possum genotype dominates. Of these three species/genotypes, only C. fayeri has been identified in two humans and the zoonotic risk is considered low. Generally, oocyst shedding in marsupials is low, further supporting a low transmission risk. However, there is some evidence of spill-back of C. hominis into kangaroo populations, which requires continued monitoring. Although C. hominis does not appear to be established in small marsupials like possums, comprehensive screening and analysis are essential for a better understanding of the prevalence and potential establishment of zoonotic Cryptosporidium species in small marsupials. Both host-specific and zoonotic Giardia species have been identified in marsupials. The dominance of zoonotic G. duodenalis assemblages A and B in marsupials may result from spill-back from livestock and humans and it is not yet understood if these are transient or established infections. Future studies using multilocus typing tools and whole-genome sequencing are required for a better understanding of the zoonotic risk from Giardia infections in marsupials. Moreover, much more extensive screening of a wider range of marsupial species, particularly in peri-urban areas, is required to provide a clearer understanding of the zoonotic risk of Cryptosporidium and Giardia in marsupials.

Rodents represent the single largest group within mammals and host a diverse array of zoonotic pathogens. Urbanisation impacts wild mammals, including rodents, leading to habitat loss but also providing new resources. Urban-adapted (synanthropic) rodents, such as the brown rat (R. norvegicus), black rat (R. rattus), and house mouse (Mus musculus), have long successfully adapted to living close to humans and are known carriers of zoonotic pathogens. Two important enteric, zoonotic protozoan parasites, carried by rodents, include Cryptosporidium and Giardia. Their environmental stages (oocysts/cysts), released in faeces, can contaminate surface and wastewaters, are resistant to common drinking water disinfectants and can cause water-borne related gastritis outbreaks. At least 48 species of Cryptosporidium have been described, with C. hominis and C. parvum responsible for the majority of human infections, while Giardia duodenalis assemblages A and B are the main human-infectious assemblages. Molecular characterisation is crucial to assess the public health risk linked to rodent-related water contamination due to morphological overlap between species. This review explores the global molecular diversity of these parasites in rodents, with a focus on evaluating the zoonotic risk from contamination of water and wasterwater with Cryptosporidium and Giardia oocysts/cysts from synanthropic rodents. Analysis indicates that while zoonotic Cryptosporidium and Giardia are prevalent in farmed and pet rodents, host-specific Cryptosporidium and Giardia species dominate in urban adapted rodents, and therefore the risks posed by these rodents in the transmission of zoonotic Cryptosporidium and Giardia are relatively low. Many knowledge gaps remain however, and therefore understanding the intricate dynamics of these parasites in rodent populations is essential for managing their impact on human health and water quality. This knowledge can inform strategies to reduce disease transmission and ensure safe drinking water in urban and peri‑urban areas.

Rabbits are highly abundant in many countries and can serve as reservoirs of diseases for a diversity of pathogens including the enteric protozoan parasites, Cryptosporidium and Giardia. Both parasites shed environmentally robust environmental stages (oo/cysts) and have been responsible for numerous waterborne outbreaks of diseases. Cryptosporidium hominis and C. parvum are responsible for most infections in humans, while Giardia duodenalis assemblages A and B, cause most human cases of giardiasis. Cryptosporidium cuniculus, the dominant species infecting rabbits, is the only spceies other than C. hominis and C. parvum to have caused a waterborne outbreak of gastritis, which occurred in the United Kingdom in 2008. This review examines the prevalence of Cryptosporidium and Giardia species in rabbits to better understand the public health risks of contamination of water sources with Cryptosporidium and Giardia oo/cysts from rabbits. Despite the abundance of C. cuniculus in rabbits, reports in humans are relatively rare, with the exception of the United Kingdom and New Zealand, and reports of C. cuniculus in humans from the United Kingdom have declined substantially since the 2008 outbreak. Subtyping of C. cuniculus has supported the potential for zoonotic transmission. Relatively few studies have been conducted on Giardia, but assemblage B dominates. However, improved typing methods are required to better understand the transmission dynamics of Giardia assemblages in rabbits. Similarly, it is not well understood if pet rabbits or contaminated water are the main source of C. cuniculus infections in humans. Well-planned studies using high-resolution typing tools are required to understand the transmission dynamics better and quantify the public health risk of Cryptosporidium and Giardia from rabbits.

The zoonotic potential of the protist parasites Cryptosporidium spp. and Giardia duodenalis in amphibians and reptiles raises public health concerns due to their growing popularity as pets. This review examines the prevalence and diversity of these parasites in wild and captive amphibians and reptiles to better understand the zoonotic risk. Research on Giardia in both groups is limited, and zoonotic forms of Cryptosporidium or Giardia have not been reported in amphibians. Host-adapted Cryptosporidium species dominate in reptiles, albeit some reptiles have been found to carry zoonotic (C. hominis and C. parvum) and rodent-associated (C. tyzzeri, C. muris and C. andersoni) species, primarily through mechanical carriage. Similarly, the limited reports of Giardia duodenalis (assemblages A, B and E) in reptiles may also be due to mechanical carriage. Thus, the available evidence indicates minimal zoonotic risk associated with these organisms in wild and captive frogs and reptiles. The exact transmission routes for these infections within reptile populations remain poorly understood, particularly regarding the importance of mechanical carriage. Although the risk appears minimal, continued research and surveillance efforts are necessary to gain a more comprehensive understanding of the transmission dynamics and ultimately improve our ability to safeguard human and animal health.

Contamination by wastewater has been traditionally assessed by measuring faecal coliforms, such as E. coli and entereococci. However, using micropollutants to track wastewater input is gaining interest. In this study, we identified nine micropollutant indicators that could be used to characterize water quality and wastewater treatment efficiency in pond-based wastewater treatment plants (WWTPs) of varying configuration. Of 232 micropollutants tested, nine micropollutants were detected in treated wastewater at concentrations and frequencies suitable to be considered as indicators for treated wastewater. The nine indicators were then classified as stable (carbamazepine, sucralose, benzotriazole, 4+5-methylbenzotriazole), labile (atorvastatin, naproxen, galaxolide) or intermediate/uncertain (gemfibrozil, tris(chloropropyl)phosphate isomers) based on observed removals in the pond-based WWTPs and correlations between micropollutant and dissolved organic carbon removal. The utility of the selected indicators was evaluated by assessing the wastewater quality in different stages of wastewater treatment in three pond-based WWTPs, as well as selected groundwater bores near one WWTP, where treated wastewater was used to irrigate a nearby golf course. Ratios of labile to stable indicators provided insight into the treatment efficiency of different facultative and maturation ponds and highlighted the seasonal variability in treatment efficiency for some pond-based WWTPs. Additionally, indicator ratios of labile to stable indicators identified potential unintended release of untreated wastewater to groundwater, even with the presence of micropollutants in other groundwater bores related to approved reuse of treated wastewater.