Overview of a long-term molecular survey in protected catchments
Maintaining safe drinking water from natural catchments requires understanding protozoan pathogens in wildlife. This longitudinal study analyzes Cryptosporidium and Giardia from 8,695 fecal DNA samples collected across 16 years (2009–2024) in Melbourne, Australia’s protected catchments. Using PCR-based sequencing and phylogenetic analyses, researchers identified 12 Cryptosporidium species/genotypes and detected Giardia duodenalis sub-assemblages AI and AIII at low prevalence, with several novel SSU sequences reported. The effort provides a robust baseline for risk management in a major urban drinking-water system.
Cryptosporidium diversity and host associations
From 8,695 samples, 274 SSU amplicons yielded 13 Cryptosporidium species/genotypes. Of these, 23 novel SSU sequences (<100% identity to GenBank) expanded the known diversity. The majority fell into three dominant clades: C. ryanae, C. fayeri, and C. macropodum, with additional sequences external to these groups. Host-by-host findings reveal complex patterns of infection across deer, marsupials, canids, lagomorphs, rodents, and birds.
Deer and other wildlife
In deer, four SSU variants represented C. ryanae and three as the deer genotype. The deer genotype v3 was most prevalent, detected in 35 of 1774 deer samples (predominantly sambar deer). Other noteworthy detections include C. muris in sambar deer and single instances of C. ubiquitum and C. occultus in deer within specific catchments. The presence of a C. parvum-like sequence in a wallaby underscores occasional cross-species signals, though gp60 amplification was unsuccessful for that sample.
Marsupials: macropods and mates
Among macropods, C. macropodum dominated, with 5 variants detected in 6014 kangaroo samples (mostly eastern grey kangaroos). A wallaby-specific C. macropodum v2 variant was found in a subset of wallabies, suggesting host-restricted subtypes within macropod populations. C. fayeri and C. ubiquitum also contributed to marsupial diversity, reflecting a rich cryptosporidial ecosystem within kangaroo hosts.
Other animals
Cryptosporidium species appeared in canids, lagomorphs, rodents, and birds. Notably, two C. canis detections occurred in a fox and a dingo. Rabbit samples yielded C. cuniculus with gp60 subtypes VbA31R4 and VbA15. Four C. baileyi detections were found in waterbirds, expanding the known avian involvement in catchment protozoa.
Giardia findings
Giardia was detected in 14 of the 8,695 samples. Giardia duodenalis sub-assemblages AI and AIII were observed: AI in deer (five individuals, including four novel AI variants) and eastern grey kangaroos (five individuals) and AI in one dog. No Giardia was detected in rabbits, rodents, or birds beyond these findings. The zoonotic potential of AI is acknowledged, though overall Giardia prevalence remained low, limiting broader conclusions.
Temporal and seasonal patterns
Cryptosporidium prevalence averaged 3.15% (274 records among 8,695 samples), rising from 0.99% in 2016 to a peak of 6.15% in 2023. Across 2009–2024, overall prevalence was 2.67% (399/14,960). Seasons influenced prevalence: spring 2.24%, summer 3.57%, autumn 2.48%, and winter 2.47%. Host groups showed distinct seasonal trends: macropods peaked in summer and spring, while deer peaked in autumn and winter. Among three dominant species (C. fayeri, C. macropodum, C. ryanae), C. macropodum peaked in summer, C. fayeri in winter/spring, and C. ryanae in autumn/winter, aligning with known birthing/calving cycles.
Implications for water safety and public health
Although Melbourne Water’s chlorination targets bacteria and viruses, it is less effective against protozoan parasites like Cryptosporidium and Giardia. The study’s long-term, large-scale molecular surveillance offers a crucial evidence base for risk assessment and management in protected catchments. Despite the detection of zoonotic species and subtypes, the absence of significant human-pathogenic Cryptosporidium species Hominis and Parvum suggests a relatively low direct human risk in these closed catchments. Nevertheless, several non-human-adapted species may carry zoonotic potential, warranting ongoing monitoring and a cautious interpretation of cross-species transmission potential.
Comparisons and context
Compared with Sydney’s catchments, Melbourne’s closed system shows markedly different species compositions, with fewer human-associated Cryptosporidium clades. Large sample sizes and catchment management practices likely influence these differences. The study also notes regional variability across Australia and emphasizes that open catchments may exhibit different protozoan profiles due to livestock and wildlife interactions.
Future directions
Further work, including gp60 and additional molecular markers, could clarify host range, zoonotic potential, and the mechanisms behind high marsupial diversity. Expanded sampling in underrepresented species and catchments will help refine risk models and support water safety planning for urban populations.
