Long-term molecular surveillance of Cryptosporidium and Giardia in wildlife in protected drinking water catchments.

BACKGROUND: This study presents findings from a 15-year longitudinal surveillance program (2009-2024) monitoring Cryptosporidium and Giardia in protected drinking water catchments in Melbourne and environs in the State of Victoria, Australia. As one of the few major cities worldwide sourcing largely unfiltered water from forested catchments, Melbourne presents a unique opportunity to assess the occurrence and prevalence of protozoan parasites in a minimally disturbed ecosystem. METHODS: A total of 14,960 animal faecal samples were analysed using polymerase chain reaction (PCR)-based sequencing, including 8695 samples collected over the past 9 years. RESULTS: Cryptosporidium was detected in 3.15% of samples and Giardia in 0.16%. A total of 12 recognised Cryptosporidium species and genotypes were identified, nine of which have known zoonotic potential, as well as two sub-assemblages (AI and AIII) of Giardia duodenalis, including four novel assemblage AI variants. Parasite diversity was the highest in eastern grey kangaroos, which hosted at least 18 Cryptosporidium variants. Temporal analyses revealed significant inter-annual variation, with peak prevalence during the 2023 La Niña year and seasonal differences by host group. Notably, C. ubiquitum, C. muris and C. occultus were recorded for the first time in these catchments. In spite of the low prevalence of high-risk species such as C. parvum and the absence of C. hominis, the detection of emerging and previously uncharacterised genotypes emphasises the importance of sustained surveillance. CONCLUSIONS: These findings have broad implications for managing zoonotic risk in unfiltered water systems worldwide. Advances in metagenomics and high-throughput sequencing platforms will be critical for enhancing future pathogen monitoring and catchment management strategies in the context of increasing climate and environmental pressures.