Molecular characterization of Blastocystis sp., Cryptosporidium spp., and Giardia spp. in Eld’s deer (Rucervus eldii) and their forest rangers in Hainan, China

BACKGROUND: Blastocystis sp., Cryptosporidium spp., and Giardia spp. are significant zoonotic enteric protozoans colonizing/parasitizing humans and animals worldwide. Eld's deer (Rucervus eldii) is a globally endangered tropical deer species. Prevalence and genetic diversity of these pathogens in wild Eld's deer and occupationally exposed human populations remain limited. This study investigated the occurrence, molecular characteristics, and zoonotic potential of these three protozoans in wild Eld's deer and their forest rangers in Hainan, China. METHODS: A total of 217 fresh fecal samples were collected from wild Eld's deer across two isolated habitats, and 19 stool samples were obtained from forest rangers, within the Hainan Bangxi Provincial Nature Reserve from March to August 2021. Genomic DNA was extracted and examined by polymerase chain reaction (PCR) targeting the SSU rRNA region for Blastocystis sp., and by nested PCR targeting the SSU rRNA region for Cryptosporidium spp., and the bg and gdh region for Giardia spp. The subtypes, species, and genotypes of positive amplicons were determined through sequence homology analysis and phylogenetic reconstruction using the neighbor-joining method. RESULTS: The overall prevalence of Blastocystis sp., Cryptosporidium spp., and Giardia spp. in wild Eld's deer were 25.8% (56/217), 5.5% (12/217), and 2.8% (6/217), respectively. Co-infections were detected in 3.2% (7/217) of deer samples. In Eld's deer, six Blastocystis subtypes were identified (ST10, ST21, ST23, ST24, ST25, ST26), with ST10 being predominant (57.1%). Five Cryptosporidium species/genotypes were characterized (Cryptosporidium bovis, Cryptosporidium xiaoi, Cryptosporidium sp. bamboo rat genotype I, Cryptosporidium sp. bamboo rat genotype III, and Cryptosporidium sp. hamster genotype), with C. bovis being predominant (50.0%). All Giardia spp.-positive samples were identified as Giardia bovis (previously known as assemblage E). In the forest rangers, one Blastocystis ST3, two C. xiaoi, and one Cryptosporidium sp. hamster genotype were detected. Notably, the isolates of Cryptosporidium sp. hamster genotype in human and wild Eld's deer showed 100% homology based on SSU ribosomal RNA (rRNA) gene sequencing analysis. Three novel sequences within Blastocystis subtypes (ST10 and ST26), three novel Cryptosporidium sequences (Cryptosporidium sp. bamboo rat genotype I, Cryptosporidium sp. bamboo rat genotype III, and Cryptosporidium sp. hamster genotype), and five novel G. bovis sequences (based on bg and gdh genes) were identified. CONCLUSIONS: This first molecular survey reveals high genetic diversity of Blastocystis sp., Cryptosporidium spp., and Giardia spp. in wild Eld's deer in China, including the detection of shared Cryptosporidium species/genotypes between this endangered deer and sympatric forest rangers. These findings suggest potential zoonotic transmission at the wildlife-human interface and underscore the need for an integrated One Health surveillance strategy in protected areas to mitigate cross-species transmission risks, thereby contributing to both wildlife conservation and public health protection.