Molecular Detection and Distribution of Giardia duodenalis and Cryptosporidium spp. Infections in Wild and Domestic Animals in Portugal

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.