Overview of the study
Maintaining safe drinking water from protected catchments requires understanding the protozoan parasites that can bypass conventional water treatment. This long-running molecular surveillance project investigates Cryptosporidium and Giardia in wildlife across Melbourne’s catchments, applying SSU-based sequencing to identify species and genotypes directly from faecal DNA samples. Between 2009 and 2024, researchers analyzed 8,695 samples, finding a Cryptosporidium prevalence of 3.15% (274 SSU amplicons) and a Giardia prevalence of 0.16%. The work provides a comprehensive, longitudinal baseline for protozoan populations in a large, closed water supply system and informs risk management for water utilities.
Methods and genetic insights
Using PCR-based sequencing and phylogenetic analysis of the small subunit rRNA (SSU) gene, 35 unique Cryptosporidium SSU sequences were assigned GenBank accession numbers, with 23 novel sequences identified (not previously published). Species and genotypes were designated based on their position in phylogenetic trees and alignment, revealing three major clades: C. ryanae, C. fayeri, and C. macropodum. The study linked each sequence to a host and catchment area, enabling spatial and host-specific interpretations of prevalence and diversity.
Cryptosporidium by host group
Deer
In the C. ryanae clade, four SSU variants represented C. ryanae and three represented the deer genotype. A total of 66 detections were recorded from 1,774 deer samples, with sambar deer contributing 49 detections. Other deer included red and fallow deer. C. muris, C. ubiquitum and C. occultus were detected infrequently, illustrating a mosaic of cryptosporidia associated with different deer species and habitats.
Marsupials
Among macropods, 116 samples yielded 5 variants in the C. macropodum clade and 13 variants in C. fayeri. Eastern grey kangaroos dominated, representing 94.1% of macropod samples. Notably, C. ubiquitum appeared in a kangaroo, marking a first macropod record, while a C. parvum-like sequence emerged in a wallaby, underscoring potential host switching or exposure to contaminated environments. A diverse array of marsupial-associated genotypes highlights the complex cryptosporidial ecology within protected habitats.
Other animals
Cryptosporidium was detected in canids (two C. canis), rodents (C. viatorum and a C. occultus variant), rabbits (C. cuniculus), and waterbirds (C. baileyi). Interestingly, four C. baileyi detections in waterbirds broaden our understanding of avian-associated cryptosporidia in water catchments.
Giardia findings
Giardia duodenalis was found in 14 samples, with deer contributing six AIII sub-assemblages and one AI. Eastern grey kangaroos harbored AI within five individuals, including four novel variants (GenBank accessions PV665481–PV665484). A single canine sample carried AI, while no Giardia was detected in rabbits, rodents (beyond those noted), or birds, limiting broader conclusions about Giardia dynamics in these groups.
Temporal and seasonal patterns
Cryptosporidium prevalence fluctuated from a low of 0.99% (2016) to a high of 6.15% (2023), with an overall mean of 3.15%. Analysis across 2009–2024 shows a significant, gradual rise in prevalence (P = 0.0175). Seasonal trends reveal higher summer prevalence for overall Cryptosporidium, while deer peak in autumn and winter and macropods peak in summer. Within the dominant species, C. macropodum peaks in January (summer) and declines in June, whereas C. fayeri and C. ryanae show winter and autumn peaks, respectively. These patterns likely reflect host biology (births and calving) and environmental drivers.
Implications for water safety
Melbourne Water’s chlorination reduces bacteria and viruses but is not designed to remove key protozoa like Cryptosporidium and Giardia. The ongoing molecular surveillance provides essential data to understand protozoan populations in catchments and to guide risk management strategies. Despite low overall Giardia prevalence and the scarce detection of C. hominis and C. parvum, the presence of several genotypes with zoonotic potential warrants continued monitoring, particularly in catchments with changing wildlife composition or hydrological conditions.
Comparisons with other catchments
Compared with Sydney’s more open catchments, Melbourne’s closed system shows far fewer hominis/parvum detections, with marsupial-associated species predominating. The contrasts emphasize the influence of land use, livestock presence, and human activity on cryptosporidial diversity in drinking water sources.
Conclusion
This long-term study represents one of the largest and most comprehensive surveillance efforts for Cryptosporidium and Giardia in wildlife within protected drinking water catchments. The integration of SSU sequencing, host information, and catchment context enables nuanced risk assessment, supports targeted management actions, and advances our understanding of protozoan ecology in Australian water systems. Ongoing surveillance remains critical to identify emerging genotypes, assess zoonotic risk, and ensure the continued safety of metropolitan drinking water.
