Detection and Molecular Characterization of Giardia and Cryptosporidium spp. Circulating in Wild Small Mammals from Portugal

SIMPLE SUMMARY: Wild small mammals can be a veterinary and public health concern, because they can act as reservoir hosts for numerous pathogens and potentially transmit them to humans, domestic animals and other wildlife species. This study represents the first investigation of the diarrhea-causing parasites Cryptosporidium spp. and Giardia spp. in wild rodents and shrews from Portugal. Cryptosporidium spp. was rarely and Giardia was frequently detected in the feces of the analyzed species, with the southwestern water voles (Arvicola sapidus) and Lusitanian pine voles (Microtus lusitanicus) showing the highest infection rates of Giardia spp. Genetic characterization based on common genomic marker sequences revealed the rodent-adapted Giardia microti and potentially zoonotic Cryptosporidium muris as the only circulating species. These findings suggest the limited role of wild rodents and shrews as natural sources of human infections in Portugal regarding the investigated parasites. Moreover, the host ranges of Giardia and Cryptosporidium spp. were extended and the obtained genetic sequences of Giardia microti are useful for future comparative studies. From the One-Heath perspective, this study helps to understand the epidemiology of Giardia spp. and Cryptosporidium spp. in wildlife. ABSTRACT: Cryptosporidium spp. and Giardia spp. are important diarrhea-causing protozoan parasites worldwide that exhibit broad host ranges. Wild small mammals can harbor host-adapted and potentially zoonotic species of both parasites. The aim of this study was to investigate Cryptosporidium spp. and Giardia spp. in wild rodents and shrews in Portugal, focusing on the protist’s occurrence and genetic diversity. Molecular screening by PCR at the small subunit (SSU) rRNA gene locus of 290 fecal samples from wood mice (Apodemus sylvaticus), southwestern water voles (Arvicola sapidus), Cabrera’s voles (Microtus cabrerae), Lusitanian pine voles (Microtus lusitanicus), Algerian mice (Mus spretus) and greater white-toothed shrews (Crocidura russula) in Northeast Portugal revealed the low occurrence of Cryptosporidium spp. (1%) and high occurrence of Giardia spp. (32.8%). The analysis revealed that “species” was the only significant factor associated with the increasing probability of Giardia spp. infection, with the highest prevalence reported in southwestern water voles and Lusitanian pine voles. Cryptosporidium and Giardia species determination at the SSU rRNA gene locus revealed C. muris and G. microti as the only circulating species, respectively. Subtyping of the glutamate dehydrogenase (gdh) and beta-giardin (bg) genes provided evidence of the high genetic diversity within the G. microti clade. This study suggests that rodent-adapted G. microti occurs to a large extent in cricetid hosts and supports the limited role of wild rodents and shrews as natural sources of human infections in Northeast Portugal regarding the investigated parasites. Moreover, this is the first record of G. microti in southwestern water voles, Lusitanian pine voles, Algerian mice, wood mice and Cabrera’s voles and C. muris in Cabrera’s voles. Finally, this study improves the database of sequences relevant for the sequence typing of G. microti strains and provides new insights about the epidemiology of Giardia spp. and Cryptosporidium spp. in wild rodents and shrews, two parasite genera of high importance for public and animal health.