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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.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Methods<\/jats:title>\n            <jats:p>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\u00a0years.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>\n              <jats:italic>Cryptosporidium<\/jats:italic> was detected in 3.15% of samples and <jats:italic>Giardia<\/jats:italic> in 0.16%. A total of 12 recognised <jats:italic>Cryptosporidium<\/jats:italic> species and genotypes were identified, nine of which have known zoonotic potential, as well as two sub-assemblages (AI and AIII) of <jats:italic>Giardia duodenalis<\/jats:italic>, including four novel assemblage AI variants. Parasite diversity was the highest in eastern grey kangaroos, which hosted at least 18 <jats:italic>Cryptosporidium<\/jats:italic> variants. Temporal analyses revealed significant inter-annual variation, with peak prevalence during the 2023 La Ni\u00f1a year and seasonal differences by host group. Notably, <jats:italic>C. ubiquitum<\/jats:italic>, <jats:italic>C. muris<\/jats:italic> and <jats:italic>C. occultus<\/jats:italic> were recorded for the first time in these catchments. In spite of the low prevalence of high-risk species such as <jats:italic>C. parvum<\/jats:italic> and the absence of <jats:italic>C. hominis<\/jats:italic>, the detection of emerging and previously uncharacterised genotypes emphasises the importance of sustained surveillance.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusions<\/jats:title>\n            <jats:p>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.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Graphical Abstract<\/jats:title>\n          <\/jats:sec>","DOI":"10.1186\/s13071-025-07048-8","type":"journal-article","created":{"date-parts":[[2025,10,14]],"date-time":"2025-10-14T15:31:42Z","timestamp":1760455902000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Long-term molecular surveillance of Cryptosporidium and Giardia in wildlife in protected drinking water catchments"],"prefix":"10.1186","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8330-6416","authenticated-orcid":false,"given":"Anson V.","family":"Koehler","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1359-8913","authenticated-orcid":false,"given":"Tao","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-3421-0428","authenticated-orcid":false,"given":"Melita A.","family":"Stevens","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6740-2405","authenticated-orcid":false,"given":"Shane R.","family":"Haydon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4423-1690","authenticated-orcid":false,"given":"Robin B.","family":"Gasser","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,10,14]]},"reference":[{"key":"7048_CR1","unstructured":"Water UN. 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