Enteric protozoan parasites Giardia duodenalis, Cryptosporidium spp., and, to a lesser extent, the ciliate Balantioides coli are responsible for severe human and animal intestinal disorders globally. However, limited information is available on the occurrence and epidemiology of these parasites in domestic, but especially wild species in Portugal. To fill this gap of knowledge, we have investigated G. duodenalis, Cryptosporidium spp., and B. coli occurrence, distribution, genetic diversity, and zoonotic potential by analyzing 756 fecal samples from several wild carnivores (n=288), wild ungulates (n=242), and domestic species (n=226) collected across different areas of mainland Portugal. Overall, infection rates were 16.1% (122/756; 95% CI: 13.59–18.96) for G. duodenalis and 2.7% (20/756; CI: 1.62–4.06) for Cryptosporidium spp., while no ungulate sample analyzed yielded positive results for B. coli. Giardia duodenalis was found across a wide range of hosts and sampling areas, being most prevalent in the Iberian lynx (26.7%), the Iberian wolf (24.0%), and the domestic dog (23.9%). Cryptosporidium spp. was only identified in wild boar (8.4%), red fox (3.4%), Iberian lynx (3.3%), red deer (3.1%), and Iberian wolf (2.5%). Sequence analysis of G. duodenalis determined zoonotic assemblage A (subassemblage AI) in one roe deer sample, canine-specific assemblages C and D in Iberian wolf, red fox, and domestic dog, and ungulate-specific assemblage E in wild boar, sheep, cattle, and horse. Six Cryptosporidium species were identified: C. scrofarum in wild boar, C. canis in the Iberian wolf and red fox, C. ubiquitum in red deer and wild boar, C. felis in the Iberian lynx, and both C. ryanae and C. occultus in red deer. Giardia duodenalis and Cryptosporidium spp. coinfections were observed in 0.7% (5/756) of the samples. This is the first, most comprehensive, and largest molecular-based epidemiology study of its kind carried out in Portugal, covering a wide range of wild and domestic hosts and sampling areas. The detection of zoonotic Cryptosporidium spp. and G. duodenalis subassemblage AI demonstrates the role of wild and domestic host species in the transmission of these agents while representing a potential source of environmental contamination for other animals and humans.A. M. Figueiredo, D. Hipólito, and J. Fernandes were supported by a PhD grant from Fundação para a Ciência e Tecnologia (SFRH/BD/144582/2019, FRH/BD/144437/2019, PD/BD/150645/2020, respectively), cofinanced by the European Social Fund POPH-QREN program. A. Dashti is the recipient of a PFIS contract (FI20CIII/00002) funded by the Spanish Ministry of Science and Innovation and Universities. R. T. Torres and J. Carvalho were supported by a research contract (2021.00690.CEECIND and CEECIND/01428/2018, respectively) from the Fundação para a Ciência e a Tecnologia. Eduardo Ferreira is funded by national funds (OE) through FCT in the scope of the framework contract foreseen in article 23, Decree-Law 57/2016. This work was supported by Centre for Environmental and Marine Studies (CESAM) through FCT/MCTES (UIDP/50017/2020+UIDB/50017/2020+LA/P/0094/2020), and national funds, Health Institute Carlos III (ISCIII) and Spanish Ministry of Economy and Competitive ness, under project PI19CIII/00029, EcoARUn (POCI-01-0145-FEDER-030310) and WildForests (POCI-01-0145-FEDER-028204) projects, funded by FEDER, through COM PETE2020-Programa Operacional Competitividade e Internacionalização (POCI), and by national funds (OE), through FCT/MCTES, project rWILD-COA: Ecological challenges and opportunities of trophic rewilding in Côa Valley—COA/BRB/0063/2019, funded by national funds (OE), through FCT/MCTES, LIFE WolFlux (LIFE17 NAT/PT/000554), Life + Project Iberlince (LIFE10NAT/ES/570), and Life Nature and Biodiversity Lynxconnect (LIFE 19NAT/ES/001055), funded by the LIFE Programme of the European Union, the EU’s funding instrument for the environment and climate action. Additional funding was obtained by “Plano de Monitorização do Lobo Ibérico PMLDS-O–ACHLI.”S
