Introduction
Cryptosporidium spp. and Giardia duodenalis are common intestinal protozoa with zoonotic potential, affecting a broad range of domestic and wild animals. In Portugal, knowledge on their occurrence and genetic diversity remains fragmented. This study integrates new molecular results from multiple animal groups with a comprehensive review of published national data to establish an updated baseline for these parasites within a One Health framework.

Methods
A total of 805 faecal samples from 91 animal species were analysed, including domestic (n = 192 samples), captive (n = 283) and wild (n = 330) hosts. Detection was performed by modified Ziehl–Neelsen and direct immunofluorescence assays; samples that were positive or doubtful after microscopic screening were further subjected to nested PCR and sequencing targeting SSU rRNA, gp60 (for C. parvum), and gdh, bg and tpi (for G. duodenalis). Newly generated sequences were compared to GenBank references and compiled with data from 29 Portuguese studies published between 2001 and 2025.

Results
Overall, Cryptosporidium spp. were detected in 11/805 samples (1.4%) and G. duodenalis in 101/805 (12.5%). Detection rates of Cryptosporidium spp. were 3.6% (7/192) in domestic, 0.0% in captive, and 1.2% (4/330) in wild animals, while Giardia spp. occurred in 18.8% (36/192), 8.5% (24/283), and 12.4% (41/330), respectively. Molecular analysis revealed C. parvum (IIaA19G2R1 in Iberian wolf, and IIcA5G3 in hedgehogs), C. canis, and C. cuniculus. Moreover, G. duodenalis assemblages were diverse, including assemblage E in ruminants, D in canids and felids, B in rabbits, lemurs, cheetahs and suricates, F in lions, and E and B co-occurring in suricates and raccoons. The compiled dataset from available Portuguese studies included 3741 animals tested for Cryptosporidium spp. and 3353 for Giardia spp., providing a descriptive overview of host and genotype diversity across the country.

Conclusions
This study provides the first integrated overview of Cryptosporidium spp. and G. duodenalis occurrences in animals across the Portuguese territory. The findings demonstrate that livestock, pets, zoo animals, and free-ranging wildlife can contribute to the circulation of these protozoan parasites, emphasizing the importance of harmonized molecular surveillance and integrated One Health strategies for early detection and control.